OBJECTIVE To explore the value of preoperative evaluation with three-dimensional endoanal ultrasonography (3D-EAUS) for anal fistula in order to provide preoperative assessment for anal fistula. METHODS One hundred patients diagnosed with anal fistula undergoing surgery between March 2012 and March 2013 in our department were prospectively enrolled. All the patients were randomly divided into the ultrasound group and the control group with fifty patients in each group. The ultrasound group received 3D-EAUS and the control group received routine examinations (digital examination and probe) to assess the position of the internal opening, the type of fistula and secondary tracks, respectively. The concordance rate of the preoperative assessment and intraoperative exploration was evaluated between the two groups. RESULTS The accuracy of identifying internal opening was 96.0% for the ultrasound group and 82.0% for the control group with statistically significant difference (P=0.02). The accuracy of identifying internal opening for simple anal fistula was similar (95.0% vs. 91.3%, P=1). For complex anal fistula, the accuracy was also higher in the ultrasound group (96.7% vs. 74.1%, P=0.025). The accuracy of fistula classification was 78.0% for the ultrasound group and 96.0% for the control group with significant difference (P=0.01). The accuracy of identifying a second track was higher in the ultrasound group (96.0% vs. 82.0%, P=0.025). CONCLUSIONS It is significantly superior for 3D-EAUS to detect the internal opening, fistula classification and identification of a second track in complex anal fistulas as compared to conventional examination. 3D-EAUS should be recommended as a preoperative assessment for anal fistula, especially for complex one.