The South African double amputee, who runs with carbon fibre prosthetic running blades after being born without a fibula in both legs, achieved the 45.25secs 'A' standard qualifying time by winning a race in Lignano.
The 24-year-old, a four-time Paralympic gold medallist, finished well clear of the field as he smashed his personal best of 45.61.
Pistorius will become the first amputee sprinter to compete at the World Championships, which get under way in Daegu, South Korea, on 27 August, and the Olympics.
Last night's race was Pistorius' final chance to seal his spot in Daegu and came just two days after he had clocked 46.65 in windy conditions in Padua.
Pistorius, who had both lower legs amputated when he was 11 months old, failed to qualify for the 400m at the 2008 Beijing Games.
In 2008, the Court of Arbitration for Sport ruled in his favour after a lengthy legal battle with the International Association of Athletics Federations over his blades.
Meanwhile, Russian pole vaulter Yelena Isinbayeva will make her Diamond League debut at the DN Galan meet in Stockholm on 29 July.
Isinbayeva took a break from competition last year and skipped the inaugural Diamond League season. The world record-holder made her season debut in Belgium last weekend, jumping 4.60 metres to win a meet in Heusden - her first outdoor competition since September 2009.
The two-time Olympic and world champion jumped 4.85 at the DN Galan in 2008.
Meet director Rajne Soderberg said "Yelena is a magnificent athlete and it's a pleasure to welcome her back."
Isinbayeva was set to compete in Lignano last night followed by a meet in Lucerne, Switzerland tomorrow as she prepares for the world championships in South Korea.