He has been charged with making false statements and obstructing a congressional investigation.
A judge scheduled the trial to begin on January 5.
Comey's lawyer, Patrick Fitzgerald, entered the plea on Comey's behalf during a roughly 25-minute court hearing on Wednesday.
"Our view is that this prosecution was brought at the direction of President Trump," Fitzgerald said.
Fitzgerald told the judge he planned to file several legal motions to dismiss the case before a trial, including filings arguing the prosecution was vindictive and that the case was tainted by "outrageous government conduct".
Fitzgerald said the defence still did not have basic information about the allegations in the indictment, including who Comey allegedly authorised to disclose information to the media.
Prosecutor Nathaniel Lemons said the case involved a "significant amount" of classified information, drawing a warning from the judge that the sensitive nature of the evidence would not set the case "off track" and delay a trial.
Comey spoke briefly in court, indicating he understood his legal rights.
His family sat in the front row of the courtroom's public gallery.
The charges were brought by Trump's former personal attorney, Lindsey Halligan, who was installed last month as the US Attorney for the Eastern District of Virginia. Trump forced out her predecessor over his reticence to prosecute Comey and New York Attorney General Letitia James.
Comey is accused of knowingly making a false statement when he told a Republican senator during a hearing in 2020 that he stood behind prior testimony that he did not authorise anyone to serve as an anonymous source in news reports about FBI investigations.
The indictment accuses Comey of authorising an FBI employee to disclose information about a federal probe.
It does not identify the investigation, but it appears to relate to Democrat Hillary Clinton, Trump's rival in the 2016 election.
It does not detail the evidence against Comey.
Comey has professed his innocence and called for a trial.
Trump has threatened to imprison his political rivals since the start of his 2016 presidential campaign, but the case against Comey marks the first time his administration has succeeded in securing a grand jury indictment against one of them.
Trump's Justice Department is also investigating other antagonists, including James, Democratic California Senator Adam Schiff, and John Bolton, who served as a national security official in Trump's first term as president.
Hours before the hearing, Trump called for jailing Chicago's mayor and Illinois' governor, both Democrats, as his administration prepared to deploy National Guard troops during stepped-up immigration enforcement in the city.
Just one in four Americans in a Reuters/Ipsos poll completed on Tuesday agreed with the statement that the Justice Department does its job fairly and without political interference.
Halligan, who has no prosecutorial experience and previously worked as an insurance attorney, presented the evidence in the case to the grand jury.
Career attorneys in the office previously drafted a memo urging her not to proceed with seeking an indictment, citing a lack of evidence to establish probable cause that he committed a crime, Reuters reported.
In a highly unusual move, the government dispatched two federal prosecutors from a different office in Raleigh, North Carolina, to handle the case.
The charges against Comey came shortly after Trump complained publicly about a lack of action on the case.
The Justice Department's willingness to respond to Trump's demands represents a breach of decades-long norms that have sought to insulate US law enforcement from political pressures.
More than 1000 Justice Department alumni from Republican and Democratic administrations recently signed a letter decrying the case against Comey as "an unprecedented assault on the rule of law".
During his first term as president, Trump fired Comey, who at the time was presiding over the FBI's investigation into contacts between Trump's 2016 presidential campaign and Russia.
The firing sparked a political firestorm and led to the appointment of Special Counsel Robert Mueller, whose investigation dogged much of Trump's first presidency.
Mueller's probe ultimately concluded there was not enough evidence to establish a criminal conspiracy.
