OBJECTIVE: To evaluate the effect of intracytoplasmic sperm injection (ICSI) using cryopreserved-thawed testicular spermatozoa with testicular fine needle aspiration (TEFNA) in patients with non-obstructive azoospermia. METHODS: Sixty-two patients underwent TEFNA. Mature testicular spermatozoa were found in 35 cases of patients and the testicular tissues were cryopreserved for later ICSI. Ovarian stimulation included a long protocol of GnRHa/FSH/hCG. Oocyte retrieval was performed under transvaginal ultrasound guidance, and ICSI conducted with cryopreserved-thawed testicular spermatozoa. RESULTS: A total of 35 couples underwent 35 ICSI cycles using cryopreserved-thawed testicular spermatozoa with testicular fine needle aspiration. The clinical pregnancy rate was 37.14% (13/35). CONCLUSION: ICSI using cryopreserved-thawed testicular spermatozoa with testicular fine needle aspiration is a main and effective method in the treatment of non-obstructive azoospermia, which can avoid further testicular fine needle aspiration.