The cDNA encoding phospholipase A₂ of Apis mellifera (AmPLA₂) was cloned into a transfer vector pFastBacHTa to form the recombinant donor plasmid pBacHT-AmPLA₂. The recombinant donor plasmid was then transformed into Escherichia coli DH10Bac. By transposition, AmPLA₂ gene was integrated into Bacmid, and a recombinant shuttle vector, rBacmid-AmPLA₂ was constructed. The cultured Trichoplusia ni Tn-5B1-4 cells, mediated with Lipofectin, were transfected with the rBacmid-AmPLA₂ DNA, and then the recombinant baculovirus, rACV-Bac-AmPLA₂ was obtained. The recombinant virus was further used to infect the Tn-5B1-4 cells to express the target protein. SDS-PAGE analysis of the infected cellular proteins showed that the size of the expression product of AmPLA₂ fused with 6×His tag at its N-terminal was about 18 kD, and the expressed protein accumulated up to about 5.35% of the total cellular proteins. Western blot analysis using anti-AmPLA₂ polyclonal serum confirmed the expressed protein was a fusion protein of AmPLA₂. The protein extracts of AmPLA₂ showed an enzymatic activity of about 6.13 μmol·min^-1·mg^-1 for hydrolyzing egg yolk substrate.