This study compared the diagnostic accuracy of percutaneous fistula contrast-enhanced ultrasound (CEUS) combined with 360° 3-D transrectal ultrasound (TRUS) imaging (CEUS+360°-TRUS) with that of conventional transrectal ultrasound in the diagnosis of complex anal fistulas. A total of 156 patients clinically diagnosed with complex anal fistula from January 2020 to December 2021 were studied and randomly divided into an experimental group (n=82) and a control group (n=74). Patients in the experimental group were examined by percutaneous fistula CEUS combined with CEUS+360°-TRUS, while patients in the control group were examined using TRUS. The detection of fistulas (main tract, branch and internal orifice) and the accuracy of Parks classification were compared between the two groups. Recurrences were followed up at 1, 3 and 6 mo after the surgery. A total of 156 patients were included, aged 23-68 y (average: 37.7 ± 18.2 y). In both groups, the course of disease was <1 mo in 128 cases, 1-2 mo in 22 cases and >3 mo in 6 cases. A total of 474 fistulas were confirmed by surgery in the aforementioned patients, including 224 main fistulas, 250 branch pipes and 254 internal orifices. The CEUS+360°-TRUS group had 96.87%, 90.41% and 90.14% diagnostic accuracy for the main tract, branch and internal orifice, which was statistically significant (p < 0.001) compared with the 85.00%, 70.00% and 72.46% for the TRUS group, respectively. The overall accuracy of Parks classification of anal fistula in the CEUS+360°-TRUS group was significantly higher than that in the TRUS group (90.24% vs. 78.38%, p < 0.001). After 6 mo of follow-up, the recurrence rate in the CEUS+360°-TRUS group was 4.87%, and the recurrence rate in the TRUS group was 18.91%. Percutaneous fistula CEUS combined with transrectal 360° 3-D imaging has significantly higher accuracy than conventional TRUS in the diagnosis of complex anal fistula, especially for anal fistula branches, internal openings and Parks classification and is beneficial in reducing post-operative occurrence of complex anal fistulas.