Professor Moriarty's first appearance and his ultimate end occurred in Doyle's story "The Final Problem", in which Holmes, on the verge of delivering a fatal blow to Moriarty's criminal ring, is forced to flee to the Continent to escape Moriarty's retribution. Moriarty follows, and the two apparently fall to their deaths while locked in mortal combat atop the Reichenbach Falls. During this story, Moriarty is something of a Mafia Godfather; he protects nearly all of the criminals of England in exchange for their obedience and a share in their profits. Holmes, by his own account, was originally led to Moriarty by the suggestion that many of the crimes he perceived were not the spontaneous work of random criminals, but the machinations of a vast and subtle criminal ring.

Moriarty plays a direct role in only one other of Doyle's Holmes stories: The Valley of Fear, which was set before "The Final Problem," but published afterwards. In The Valley of Fear, Holmes attempts to prevent Moriarty's agents from committing a murder. Moriarty does not meet Holmes in this story. In an episode where Moriarty is interviewed by a policeman, a painting by Jean-Baptiste Greuze is described as hanging on the wall; Holmes remarks on another work by the same painter to show it could not have been purchased on a professor's salary. The work referred to is La jeune fille à l'agneau; some commentators have described this as a pun by Doyle upon the name of Thomas Agnew of the gallery Thomas Agnew and Sons, who had a famous painting stolen by Adam Worth, but was unable to prove the fact.

Holmes mentions Moriarty reminiscently in five other stories: "The Empty House" (the immediate sequel to "The Final Problem"), "The Norwood Builder," "The Missing Three-Quarter," "The Illustrious Client,", and "His Last Bow." More obliquely, a 1908 mystery by Doyle, The Lost Special, features a criminal genius who could be Moriarty and a detective who could be Holmes, although neither is mentioned by name.

Although Moriarty appeared in only two of the 60 Sherlock Holmes tales by Conan Doyle, Holmes' attitude to him has gained him the popular impression of being Holmes' arch-nemesis -- as "The Final Problem" clearly states, Holmes spent months in a private war against Moriarty's criminal operations—and he has been frequently used in later stories by other authors, parodies, and in other media.

In the Doyle stories, narrated by Holmes' assistant Dr. Watson, Watson never meets Moriarty (only getting distant glimpses of him in "The Final Problem"), and relies upon Holmes to relate accounts of the detective's battle with the criminal.

Doyle himself is inconsistent on Watson's familiarity with Moriarty. In "The Final Problem", Watson tells Holmes he has never heard of Moriarty, while in The Valley of Fear, set earlier on, Watson already knows of him as "the famous scientific criminal."

Moriarty's weapon of choice is the "air-rifle", a unique weapon constructed for the Professor by a blind German mechanic, von Herder, and used by his employee Colonel Sebastian Moran. It closely resembled a cane, allowing for easy concealment, was capable of firing revolver bullets and made very little noise when fired, making it ideal for sniping; the weapon became infamous for being Moriarty's favorite tool. Moriarty also has a marked preference for organising "accidents". His attempts to kill Holmes include falling masonry and a speeding horse drawn van. He is also responsible for stage managing the death of Birdy Edwards.

Holmes described Moriarty as follows:

But the man had hereditary tendencies of the most diabolical kind. A criminal strain ran in his blood, which, instead of being modified, was increased and rendered infinitely more dangerous by his extraordinary mental powers. Dark rumours gathered round him in the University town, and eventually he was compelled to resign his chair and come down to London. He is the Napoleon of Crime, Watson, the organiser of half that is evil and nearly all that is undetected in this great city..."

The "smaller university" involved has been claimed to be one of the colleges that later comprised the University of Leeds. However, in Sherlock Holmes: The Unauthorized Biography, the "smaller university" is said to be Durham.