Objective To verify the technics of inactivating/removing virus in collagen sponge derived from bovine Achilles tendon. Methods Possible pathogen species were determined according to the raw material of bovine Achilles tendon used in production, then vesicular stomatitis virus (VSV), theiler’s mouse encephalomyelitis virus (TEMV), pseudorabies virus (PRV), and simian vacuolating virus 40 (SV40) were selected as indicator virus. Virus suspension was prepared in accordance with Technical Standard for Disinfection. 60Co radiation 25 kGy of collagen sponge was determined as inactivating/removing virus process according to the analysis of the manufacture process, the virus inactivation/removal effect was verified by the measurement of median tissue culture infective dose (TCID50) and showed by virus reduction factor (sample average values of numerical difference before and after processing). Results Reduction factors of VSV, TEMV, PRV, and SV40 after 60Co radiation 25 kGy were 5.646, 4.792, 5.042, and 5.292 logTCID50/0.1 mL (logs), respectively. Reduction factor of each indicator virus was greater than 4 logs, showing that 60Co irradiation 25 kGy can effectively inactivate and remove viruses. Conclusion 60Co radiation 25 kGy of collagen sponge derived from bovine Achilles tendon can be used as the technics of inactivating/removing virus during the preparation process of collagen sponge to guarantee the safety of the product.