Objective To reduce experimental costs and improve the utilization of S9,we use spiral coating technique to evaluate the activity of rat liver S9 prepared by combination inducing method as well as to establish cryopreservation method. Methods Using spiral coating technique and Ames test to evaluate the activity of self-made rat liver S9 and commercially available S9 separately. We use glycerinum as protective agent to establish cryogenic storage method, so that S9 can be in liquid form stored at -20 ℃ in the refrigerator. Results In the Ames assay as well as using spiral coating technique, the number of revertant colonies had dose-response relationship among the dose of S9. When conditions were the same, the number of revertant colonies in positive control was at the approximate level in presense of self-made rat liver S9 and commercially available S9 respectively. When S9 (concentration of 38%) was added to the amount at 1.48～6.62 μL/μL broth dose of bacteria, it can significantly induced Salmonella typhimurium histidine strains TA100, reverse mutation rates were three times more than the control group. Conclusions Spiral coating technique can successfully evaluate the activity of rat liver S9. The inducing method of combination of PB and BF can take the place of the unducing method of PCB_s in the preparation of Liver homogenate S9.