Objective: To explore the application of transperineal three-dimensional pelvic floor ultrasound in the diagnosis and evaluation of female stress urinary incontinence (SUI). Methods: A total of 120 patients with SUI who were treated in our hospital from July 2020 to December 2021 were selected as SUI group, and they were divided into mild group (n=28), moderate group (n=56) and severe group (n=36) according to the disease classification. Another 40 healthy women who underwent physical examination in the same period were selected as the control group. The general information of all subjects and the ultrasonographic parameters of Valsalva status [bladder neck descent (BND), urethral rotation angle, and bladder posterior angle] were collected. Univariate analysis and Pearson correlation coefficient were used to analyze the relationship between related indicators and SUI. Receiver operating characteristic (ROC) curve was used to analyze the efficacy of transperineal three-dimensional pelvic floor ultrasound in the diagnosis and evaluation of SUI. Results: The ultrasonic parameters BND, urethral rotation angle and bladder posterior angle in the SUI group were significantly higher than those in the control group (all P<0.05). The BND, urethral rotation angle and posterior bladder angle of mild group, moderate group and severe group increased in turn, and the differences between the groups were statistically significant (all P<0.05). ROC curve analysis showed that BND, urethral rotation angle and bladder posterior angle had certain value for the diagnosis and disease evaluation of female SUI, and the application value of combination of the three was the highest. Diagnostic SUI: area under the curve (AUC) was 0.991, sensitivity was 94.17%, specificity was 100.00%. Moderate SUI: AUC was 0.914, sensitivity was 92.86%, specificity was 71.43%. Evaluation of severe SUI: AUC was 0.937, sensitivity was 94.44%, specificity was 87.50%. Conclusion: Transperineal three-dimensional pelvic ultrasound has high diagnostic value for female SUI, and can be used to evaluate the severity of SUI, providing objective and scientific basis for clinical diagnosis and treatment.