Prostate cancer (PCa) is a common malignant tumor of the male urinary system and ranks the second in the causes of tumor-related deaths. Differential display code 3 (DD3) is a noncoding gene that is specifically expressed in PCa. High expression of sperm-associated antigen 9 (SPAG9) is closely related to tumorigenesis of PCa, and SPAG9 is a therapeutic target for PCa. In this study, a new oncolytic adenovirus DD3-ZD55-SPAG9 was constructed by using DD3 promoter to enhance the efficacy and safety of adenovirus. The combined use of DD3-ZD55-SPAG9 and docetaxel showed that DD3-ZD55-SPAG9 significantly improved the anti-tumor efficacy of docetaxel in PCa both in vitro and in vivo. The mechanism was related to the induction of tumor cell apoptosis and the inhibition of tumor cell invasion. In conclusion, DD3-ZD55-SPAG9 combined with docetaxel is an effective strategy for PCa therapy.