Bristol County Assistant District Attorney William McCauley wants Judge Susan Garsh to remove herself from the case, he said during a hearing in Fall River Superior Court.
McCauley did not detail his reasons in court, but a new filing cited a "well-known and publicly documented history of antagonism" between him and Garsh, stemming from a 2010 murder trial he argued before her.
Though McCauley won a conviction in that case, he was quoted in the media as criticizing Garsh, saying she had unfairly limited or excluded evidence and exhibited hostility.
The filing said the friction would likely be exploited and sensationalized by the media in the high-profile case and could impair the ability of McCauley and Garsh to perform their sworn duties.
"This isn't a matter the Commonwealth takes lightly," McCauley told the judge.
Hernandez, 23, was indicted in August in the killing of 27-year-old Odin Lloyd, a semi-professional football player who was dating the sister of Hernandez's girlfriend. He pleaded not guilty to first-degree murder and five weapons-related charges last month and is being held without bail at a county jail.
Hernandez attorney James Sultan said in court that the defence would strenuously object to the recusal request when arguments are heard Oct. 21.
Outside court, another of his attorneys, Charles Rankin, declined to comment further. "We are very, very much looking forward to Aaron's day in court," he said.
The defence has said previously that prosecutors' case won't hold up during a jury trial and they are confident Hernandez will be exonerated.
Prosecutors allege Hernandez orchestrated Lloyd's killing because he was upset with him for talking to some people at a nightclub with whom Hernandez had problems.
Messages seeking comment on the recusal request were left with Garsh. She didn't address their history in the hearing.
McCauley has previously sought Garsh's recusal in a case. In a 2011 court filing, resubmitted Wednesday in support of the new request, he wrote that she had "exhibited antagonism and bias toward the prosecution throughout the (2010) case" and that he didn't believe she could be free of bias.
During Wednesday's hour-long hearing, Hernandez briefly took the witness stand to answer a string of questions from the judge about a possible conflict of interest in the case by another of his attorneys, Michael Fee. The wife of one of the prosecutors is a partner at the firm where Fee is also a partner.
The questioning was to ensure Hernandez knew of the potential conflict and still wished to be represented by Fee. He said he did.
Four others face charges in the case. Ernest Wallace and Carlos Ortiz, who are said to have been with Hernandez and Lloyd in a car on the night Lloyd was killed, have been charged with being an accessory to murder after the fact.
According to prosecutors, Ortiz told authorities he did not see who shot Lloyd, but that Hernandez was alone with him before it happened. He initially told police a different story, saying both Hernandez and Wallace got out of the car at the industrial park in North Attleborough, Mass., where Lloyd's bullet-riddled body was found.
Wallace has pleaded not guilty. Ortiz has not yet been arraigned on the accessory charge but has pleaded not guilty to a gun charge.
Hernandez's girlfriend, Shayanna Jenkins, has been indicted on a perjury charge but not yet been arraigned.
Hernandez's cousin, Tanya Singleton, has been indicted on charges including conspiracy to commit accessory after the fact and criminal contempt. Prosecutors say she refused to testify before the grand jury hearing evidence in the case even though she was offered immunity. Singleton has pleaded not guilty to both charges.
Hernandez has also been linked to an investigation into a double homicide in Boston in 2012. While investigating Lloyd's death, police found a sport utility vehicle rented in Hernandez's name at the home of Hernandez's uncle in Bristol, Conn., that they had been wanted in connection with those killings.
Associated Press writer David Klepper contributed to this report.