In Ian Fleming's first draft of Casino Royale (1953), Moneypenny's name was originally "Miss 'Petty' Pettaval", which was taken from Kathleen Pettigrew, the personal assistant to MI6 director Stewart Menzies. Fleming changed it to be less obvious. Other candidates for Moneypenny's inspiration include Vera Atkins of Special Operations Executive; Paddy Ridsdale, a Naval Intelligence secretary; Joan Bright Astley, whom Fleming dated during World War II, and who was noted for giving a warm and friendly reception to senior officers who visited her office to view confidential papers; and Joan Howe, Fleming's red-haired secretary at The Times who had typed the manuscript of Casino Royale. The BBC has used the term "Fleming's Miss Moneypenny" when referring to Jean Frampton, who typed out the manuscripts for Fleming's later works and made plot suggestions to him, even though the two never met.

Miss Moneypenny is the private secretary of M, the head of MI6. She holds the rank of Second Officer in the Women's Royal Naval Service, which is a prerequisite rank for this position. She is cleared for Top Secret, Eyes Only, and Cabinet-Level intelligence reports, the last of which she is often required to prepare, and in some cases present.

M's personal assistant is utterly dedicated to her work, which means she has little time for a social life. A close confidante of her boss, she also enjoys a flirtatious—though never consummated—relationship with James Bond, whom she understands perfectly.

Moneypenny was never given any backstory until the film Skyfall (2012), when she was re-introduced to the series following the 2006 reboot of the series' continuity. Moneypenny, now played by Naomie Harris and given the first name Eve, is originally a field officer assigned to work with Bond on an operation in Istanbul. It ends in disaster when she is ordered to shoot through Bond while he is fighting hand-to-hand combat with the mercenary they are chasing. She hits Bond, who falls off a bridge and is assumed dead. She is temporarily suspended for this and reassigned to desk duty, assisting Gareth Mallory (Ralph Fiennes), the Chairman of the Intelligence and Security Committee, who has been assigned to watch over MI6. When Bond resurfaces and returns to duty, she meets with him in Macau and aids in locating an agent of main villain Raoul Silva's (Javier Bardem) before returning to London. Later in the film, she is a participant in another shootout with Silva, when he ambushes a public inquiry into MI6. By the end of the film, she decides to retire from fieldwork and becomes Mallory's secretary once he takes over the role of M.

In both the Bond novels and films based upon them, Moneypenny is smitten with Bond. For example, in the novel Thunderball (1961), Fleming wrote that she "often dreamed hopelessly about Bond." However, she never explicitly voices these feelings.

Miss Moneypenny's role in Fleming's novels is even smaller than her role in the films. In the novels, Bond also has his own secretary, Loelia Ponsonby and later Mary Goodnight, both of whose lines and relationships were often transferred to Miss Moneypenny for the films. As a rule, Moneypenny generally never directly participates in Bond's missions. However, in Skyfall, Moneypenny is an MI6 officer who directly assists Bond in the field before becoming the new M's secretary. In the film Octopussy, Moneypenny has an assistant named Penelope Smallbone (Michaela Clavell), who appears to be equally smitten with Bond, despite a "thorough briefing" on the subject by Moneypenny. Intended as either a foil or a replacement for Moneypenny, Smallbone appeared only that once.

In most of the Bond films, there is a scene, usually Bond's arrival at M's office, in which Bond and Moneypenny exchange witty, flirtatious conversation. ("Flattery will get you nowhere, but don't stop trying.") In the earlier films, these exchanges are more sexually charged, with Bond often kissing or caressing Moneypenny sensually. In Die Another Day (2002), she puts on Q's (John Cleese) virtual reality glasses and runs a simulation in which she and Bond finally consummate their relationship. Q interrupts her, and she pretends she was using it as a combat simulation.

In the original film version of Casino Royale, actress Barbara Bouchet plays M's current secretary and explains to Sir James Bond (played by David Niven) upon their first meeting that she is actually Miss Moneypenny's daughter. She is referred to thereafter, and in the closing credits, as Moneypenny.

Since the character's first appearance in Casino Royale, neither Fleming nor any succeeding Bond novelist gave Moneypenny a first name. In a number of books and at least one film, Bond refers to her by the nickname "Penny" (a shortened version of her last name). However, The Moneypenny Diaries gives her first name as Jane, while in Skyfall, the character is named Eve.

After Lois Maxwell's death, Roger Moore recalled that she would have liked to have become the new M after Moore's retirement. She had suggested herself for the role of M but had been turned down. "I think it was a great disappointment to her that she had not been promoted to play M. She would have been a wonderful M."