Pistorius, who competes wearing carbon fibre prosthetic blades, needed to run the A qualifying time of 45.30 seconds but placed second in the 400 final in a time of 45.52.
South Africa's Sports Confederation and Olympic Committee had set June 30 as the cut-off point for athletes to qualify for the Games so the African championships had been slated as Pistorius' last chance to ensure his place.
"Thank you to everyone for your support! I came 2nd in the final of the African championships with a 45.52s. Was a rough week and I'm blessed," Pistorius, who was chasing his dream of becoming the first double-amputee to compete on the track at an Olympics, said on his Twitter account.
The 25-year-old achieved the 'A' qualifying time when he clocked 45.20 in March but Athletics South Africa (ASA) require their athletes to go under the time twice, with one of them being at an international meet.
Pistorius became the first amputee athlete to compete at the world championships last year in Daegu and his hopes of competing at the Olympics may not be completely over as he could still be picked in the 4x100 relay team.
ASA President James Evans told Reuters in a telephone interview that the South African relay teams would probably be announced on Monday (July 2) while playing down the significance of Pistorius missing out on automatic Olympic qualification.
"I think the bigger story is that Oscar has won a silver medal at a continental championships," he said.
The London Olympics start on July 27.
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