Investigators revisited the basement of the building, located half a block away from the home of Etan Patz, who vanished May 25, 1979, and found possible traces of blood on a wall, CNN reported.
Agents sprayed the wall with luminol, the chemical widely seen in television crime shows that reacts with blood residue by giving off a blue glow.
Parts of the wall were removed and sent to an FBI laboratory in Virginia for analysis, officials who asked not to be identified told CNN.
The basement in an apartment building that once housed a restaurant, is where Othniel Miller, a now-retired 75-year-old handyman, had a workshop when Patz disappeared. Miller, who now lives in Brooklyn, has been questioned several times and has repeatedly denied any knowledge of what happened to the boy in Manhattan's SoHo neighborhood.
The initial investigation reported the basement's concrete floor had been newly poured, although it was never dug up. Investigators were spending the weekend digging up the basement, the New York Daily News said.
Patz disappeared on the first day his parents allowed him to walk to a bus stop to go to school.
He was legally declared dead in 2001.