The APN3 gene from the midgut of Plutella xylostella（L.）was amplified using temperature gradient PCR with degenerate primers. Two weak bands were apparent after electrophoresis of the first PCR products. Therefore, a second PCR was performed with the first PCR product as the template and original primer. The optimal primer was finally selected, which can be used to amplify partial APN3 cDNA at an annealing temperature of 48℃. These results provide techniques to study the gene function of APN3 and phylogenesis among APN isoforms. After comparing electrophoresis bands we speculate that the expression of APN3 is far lower than that of APN1, APN2 and APN5. We conclude that APN3 is most closely related to APN1 and most distantly related to APN2. These results are important to understanding how Bt toxins are toxic to P. xylostella and the molecular resistance mechanism of P. xylostella to these toxins.