Abstract: In order to establish a multiplex PCR detection method of genetically modified ingredient in Populus nigra, primers were designed according to CaMV 35s promoter, NOS terminator, NPTⅡ marker gene and Bt target gene sequences of insect-resistant transgenic P. nigra. The promoter, terminator, marker gene and target gene were detected by simplex PCR and multiplex PCR. The PCR system such as annealing temperature and content of primers were optimized. The results showed that the high efficiency PCR system was obtained when the final ratio of the primer is 1.0∶ 0.5∶ 0.5 for Bt, NPTⅡ and NOS respectively, and the annealing temperature is 59 ℃. Base on these, a technical system was set up to detect the Bt, NPTⅡ, NOS gene of insect-resistant transgenic P.nigra with triple PCR, and the quick, efficient and accurate identification way of insect-resistant transgenic P.nigra was achieved.