The comparative study of different detection methods for Avian leukosis virus in poultry live vaccine were carried out using direct ELISA method，RT-PCR method，virus isolation and SPF chicken antibody detection method. Avian leukosis virus contamination was detected in live poultry vaccines of different origins. By further sequencing，it was found that both the direct ELISA assay and the RT-PCR assay were “false positive”. The test results showed that although the virus separation method was accurate，it was difficult to operate and high technology should be required. The SPF chicken antibody detection method was simple and accurate. And the purity of the vaccine could be reflected，which was conducive to popularization and application.