BACKGROUND: Surgical treatment is currently the only treatment for hypospadias, but the incidence of postoperative complications is still high, among which urethral fistula is the most common one. So, the aim of this study was to investigate the influencing factors of urethral fistula in hypospadias patients after tubularized incised plate (TIP) urethroplasty repair. METHODS: Proteomics analysis was performed to detect differences expression in proteins between groups (hypospadias without complications, hypospadias with urethral fistular). Multimodal verification of myeloperoxidase (MPO) expression was conducted through Immunohistochemistry, immunofluorescence, and western blotting. After collection of 21 cases of children with hypospadias from June 2018 to June 2020, the analysis was performed to investigate the association between MPO levels and clinical characteristics. RESULTS: The expression of MPO in foreskin of hypospadias with postoperative complications, was significantly higher than that of the foreskin tissue of the uncomplicated group. The results of immunohistochemistry analysis of 21 children showed that children with high MPO expression were more likely to develop urethral fistula after surgery. CONCLUSIONS: Our findings revealed that high expression of MPO may be closely related to the occurrence of urethral fistula in hypospadias after TIP urethroplasty repair.