Abstract: The study was carried out on isolation, purification and culture of spermatogonia gained from chicken testicular tissues. After enzymatic digestion, the single cell suspension was treated by percoll density gradient centrifugation to obtain high purity’s spermatogonia. The results showed that ①After percoll gradient centrifugation, the purity of spermatogonia gained from 6d-old-cock was 10.2%, 5.7% and 2.5% higher than the purity from 13d-old-cock, 15d chicken embryo and 19d chicken embryo. ②By further using adhesion purification, 82% of spermatogonia was also obtained, which was 15.6% higher than the purity of only using percoll density gradient centrifugation. ③Before and after percoll density gradient centrifugation, the different methods for cultivation spermatogonia in vitro were also compared. Results showed that: in the condition of no CEF feed layer existed, the adhesion time and survival time of spermatogonia before percoll density gradient centrifugation was earlier and longer than the time after percoll density gradient centrifugation.