To investigate the clinical effects of autologous split-thickness skin grafting for prefabricating urethra combined with scrotal flap in repairing middle urethral defect with penile defect. The retrospective observational study was conducted. Eight male patients (aged 14 to 58 years) with middle urethral defect and penile defect caused by various injuries who met the inclusion criteria were admitted to the First Affiliated Hospital of Air Force Medical University from January 2015 to January 2022. The length of urethral defect was 3 to 5 cm, and the wound area of penile defect after debridement was 5.0 cm×2.5 cm to 7.0 cm×5.5 cm. All the patients underwent autologous split-thickness skin grafting for prefabricating defect urethra in stage Ⅰ, and urethral anastomosis was performed and unilateral scrotal flap was transferred to reconstruct urethra and penis in stage Ⅱ. The area of scrotal flap was 6.0 cm×3.0 cm to 8.0 cm×6.0 cm. The wound in the donor area of skin graft was covered by oil gauze, and the wound of flap donor area was sutured directly. On the 7 day after the operation of stage Ⅱ, the survival of the flap was observed. In 3 weeks after the operation of stage Ⅱ, the urinary flow rate was measured by the urinary flow rate detector (urinary flow rate >15 mL/s was regarded as unobstructed urination), the urinary fistula and erectile function were observed, and the self-made therapeutic satisfaction questionnaire was used to investigate the therapeutic satisfaction degree of patients. During follow-up, the appearance of the flap recipient area was observed, the Vancouver scar scale (VSS) was used to evaluate the scar situation in the donor areas of skin graft and flap, the urinary flow rate was detected as before, the urethral stricture, urinary fistula, and erectile function were observed, and the therapeutic satisfaction degree of patients was investigated. On the 7 day after the operation of stage Ⅱ, the flaps survived completely in 8 patients. In 3 weeks after the operation of stage Ⅱ, the urinary flow rate was 25.3 (18.0, 38.5) mL/s, with unobstructed urination, without urinary fistula and with erectile function, and the score of therapeutic satisfaction degree was 14.3 (14.0, 15.0). During follow-up of 1 to 7 years, the flap recipient area of 8 patients was full in appearance and not swollen, with similar color to the surrounding tissue; the VSS scores of the donor areas of skin graft and flap were 11.5 (10.0, 13.0) and 10.5 (9.3, 12.0), respectively, the urinary flow rate was 24.6 (17.7, 34.1) mL/s, with no urethral stricture, urinary fistula, and erectile dysfunction, and the score of therapeutic satisfaction degree was 13.5 (13.3, 14.8). Autologous split-thickness skin grafting for prefabricating urethra combined with scrotal flap in repairing the urethral and penile defects not only reconstructs the structure of urethra and the shape of penis, but also restores the sensation and erectile function of penis, with few postoperative complications, no obvious scar hyperplasia, and high satisfaction degree of patients, which is worthy of clinical promotion.