Abstract: Objective To explore the clinical value of hydrogen peroxide-enhanced transanal ultrasound (HPUS) in diagnosis of anal fistulas. Methods Clinical and imaging data of 54 patients with anal fistula who were treated in our hospital from January 2016 to August 2017 were retrospectively analyzed. Accuracies of HPUS and color Doppler ultrasound (CDFI) for diagnosis of anal fistulas were evaluated using surgical finding as gold standard. Results The accuracy of HPUS was similar with CDFI in diagnosis of low simple anal fistulas (100% vs 100%), but higher for high simple anal fistula (100% vs 77.7%, P=0.081) and lower complex fistula(100% vs 78.6%, P=0.082) without significant differences. Moreover, HPUS was superior to CDFI in diagnosis of high complex fistulas (90% vs 38.1%, P< 0.01), especially for location of internal opening (94.4% vs 66.7%, P=0.014), and secondary extension(92.6% vs 59.3%, P< 0.01). There was no significant difference in diagnosis of the primary fistula tract (96.3% vs 90.7%, P=0.083)between the two methods. Conclusion HPUS can be used as an optimal choice for the diagnosis of complex anal fistulas.