The South African, who runs with carbon fibre blades attached below the knees, is entitled to compete against able-bodied runners following a Court of Arbitration for Sport ruling in May.
The 21-year-old needed to clock 45.55 to qualify for the individual race at next month's Beijing Games but instead ran 47.78 to finish fourth.
He has another chance to reach the mark at the Rome Golden Gala on July 11 and at a meeting in Lucerne five days later after Athletics South Africa gave him extra time to try to qualify.
"I am a bit disappointed. I didn't expect to be wonderful but I didn't expect to be this bad," Pistorius told reporters.
"But I have to be realistic about it. I haven't run in about a year."
The South African, winner of the 200 gold and 100 bronze at the 2004 Athens Paralympics, may still be able to run in his country's relay team in Beijing even if he does not manage the individual time.
In another standout race at the Notturna di Milano meeting at the ageing Arena, Italy's Olympic 20 km walk champion Ivano Brugnetti geared up for Beijing with victory in the men's five kms walk in 19:07.72.
Ethiopian world indoor champion Deresse Mekonnen easily won the men's 1,500 in 3:34.02 while Britain's world silver medallist Nicola Sanders triumphed in the women's 400 in 50.87.