Gerard Marrone told the New York Daily News Thursday he resigned because of "the horrific way this boy [Leiby Kletzky] was killed."
"I have three little boys," he said. "You can't look at your kids and then look at yourself in the mirror, knowing that a little boy, who's close in age to my eldest son, was murdered so brutally."
Aron, 35, who is charged with first-degree murder and faces life in prison without the possibility of parole under New York law if convicted, still has one attorney, Pierre Bazile.
A former federal prosecutor, Morris Fodeman, told the Daily News Aron could be eligible for the federal death penalty if he took 8-year-old Leiby across state lines.
Police said they believe Aron drove the boy to an upstate New York wedding, stopping on the way at a gas station on the Palisades Parkway in New Jersey.
U.S. Attorney Loretta Lynch, in the borough of Brooklyn, declined comment, but a source said her office "has no interest" in taking on the case.