The APF has yet to name a replacement for Uruguayan Pelusso, Paraguay's second coach in the qualifying campaign in which the team are bottom of the South American group with eight points from 12 matches and a goal difference of minus 14.
"(The APF) informs that coach Gerrardo Pelusso has officially left the APF as trainer of the Paraguay football team at a meeting he held with the national teams director," the APF said in a statement.
Mathematically, the 2010 quarter-finalists could sneak into the Brazil tournament with four matches still to play.
Pelusso took charge last July after Francisco Arce was sacked but he failed to improve a team whose South Africa 2010 veterans were past their best. He managed one win and one draw in seven matches.
The 59-year-old Uruguayan was appointed after two successful seasons in charge of leading Paraguayan club Olimpia including a league title in 2011.
Paraguay reached their fourth successive World Cup finals in 2010 and the 2011 Copa America final under Argentine coach Gerardo Martino who then quit with the young and inexperienced Arce taking his place.
The teams finishing in the top four places in the nine-nation South American group qualify for the 2014 finals in Brazil. The fifth-placed team will face Asia's fifth-ranked qualifier in a two-legged playoff for another spot in the tournament.