Kelly Ann Ayotte was born in Nashua, New Hampshire, on June 27, 1968, the daughter of Kathleen M. (née Sullivan) and Marc Frederick Ayotte. Her father's family is of French–Canadian descent. Ayotte attended Nashua High School and received a B.A. from Pennsylvania State University in political science. At Penn State, she was initiated into the Delta Gamma sorority. In 1993, Ayotte received a J.D. from Villanova University School of Law, where she had served as editor of the Environmental Law Journal.

Ayotte clerked for Sherman D. Horton, associate justice of the New Hampshire Supreme Court, for one year. From 1994 to 1998, she was an associate at McLane, Graf, Raulerson & Middleton, a Manchester law firm.

In 1998, Ayotte joined the office of the New Hampshire attorney general as a prosecutor. In 2001, she married Joseph Daley, a pilot in the National Guard. In 2003, Ayotte became legal counsel to Governor Craig Benson. Three months later, she returned to the attorney general's office as deputy attorney general. In June 2004, Benson appointed Ayotte as attorney general of the state of New Hampshire after Peter Heed resigned. Ayotte had both her children while serving as the first and only female New Hampshire attorney general.
Ayotte joined eight other states' attorneys general to sue federal regulators over a rules change that made clean air emissions standards for power plants less strict and eliminated clean air reporting and monitoring requirements.

In 2005, the court agreed with Ayotte and the others that the Environmental Protection Agency must measure changes in the emissions from power plants and could not exempt power plants from reporting their emissions.
As assistant attorney general, Ayotte prosecuted two defendants for the 2001 Dartmouth College murders in Etna, New Hampshire.

As attorney general, Ayotte prosecuted the high-profile case surrounding the 2006 murder of Manchester police officer Michael Briggs in the line of duty. It resulted in a conviction and death penalty sentence. Members of Briggs's family praised her leadership in ads for her 2010 Senate campaign.
In 2003, the United States District Court for the District of New Hampshire found the Parental Notification Prior to Abortion Act, a New Hampshire law requiring parental notification of a minor's abortion, unconstitutional, and enjoined its enforcement. In 2004, New Hampshire Attorney General Peter Heed appealed the ruling to the United States Court of Appeals for the First Circuit, which affirmed the district court's ruling. In 2004, Ayotte appealed the First Circuit's ruling to the Supreme Court, over the objection of incoming Governor John Lynch. She personally argued the case before the Supreme Court. The Supreme Court unanimously vacated the district court's ruling and remanded the case back to the district court, holding that it was improper for the district court to invalidate the statute completely instead of just severing its problematic portions or enjoining its unconstitutional applications. In 2007, the New Hampshire legislature repealed the law, mooting the need for rehearing by the district court.

In 2008, Planned Parenthood sued to recover its attorney fees and court costs from the New Hampshire Department of Justice. In 2009, Ayotte, as attorney …

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Ayotte resigned as attorney general on July 7, 2009, to explore a run for U.S. Senate in 2010. The crowded Republican primary field included former congressional and gubernatorial candidate Ovide M. Lamontagne, businessman and owner of NH1 News William Harrison Binnie, and State Representative Tom Alciere. Ayotte had never run for office, but narrowly won the primary election on September 14, 2010. In the general election, she defeated Democratic nominee U.S. Representative Paul Hodes by a vote of 273,218 (60%) to 167,545 (37%).
Ayotte was endorsed by John McCain, Sarah Palin, John Thune, Tom Coburn, Mitt Romney, Tim Pawlenty, Haley Barbour, and Rick Santorum. According to one senior GOP aide, "The addition of a Republican woman from New England who's young, who's a mom … all of these things broaden the Republican party's appeal and say to different segments of the population, 'This party has folks in it that are just like you.'"
In 2016, Ayotte ran for reelection to the U.S. Senate against Maggie Hassan, New Hampshire's sitting governor.

In February 2016, the Koch Brothers-linked conservative advocacy group Americans for Prosperity announced that Ayotte was the lone vulnerable Republican U.S. senator the group would not support in 2016, due to her support for the Clean Power Plan to combat climate change.

On May 4, 2016, an Ayotte spokeswoman said Ayotte "intends to support the Republican nominee" for U.S. president but did not plan to make an endorsement. In October 2016, after lewd sexual comments Republican nominee Donald Trump made in a 2005 video came to light, Ayotte said that as a mother and a former prosecutor who had worked with victims, she could no longer vote for Trump, and would write in Mike Pence for president.

Ayotte lost the election to Hassan by 1,017 votes.
Ayotte was endorsed by the New Hampshire Troopers' Association, the New England Narcotics Enforcement Officers' Association, and the Manchester Police Patrolmen's Association. She was also endorsed by the New Hampshire Union Leader, the Nashua Telegraph, the Caledonian-Record, and the Portsmouth Herald. The Herald endorsement was notable as it had endorsed Ayotte's opponent, Maggie Hassan, in Hassan's previous runs for office.
Ayotte helped include provisions in the Every Student Succeeds Act to boost STEM education, particularly among girls and underrepresented minorities, and to support career and technical education in schools.

Ayotte strongly opposed the National Oceanic and Atmospheric Administration's proposal to pass significant at-sea monitorin…

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Ayotte was named to several corporate boards of directors, including those of Caterpillar Inc., News Corp., BAE Systems, Boston Properties, Blink Health, Bloom Energy, and Blackstone Group.