Accurate preoperative evaluation of fistula-in-ano can guide the choice of surgical procedure and may improve healing rates. This prospective study aimed to evaluate the accuracy of conventional 3D transperineal ultrasound (3D-TPUS) compared with SonoVue (SVE)-enhanced 3D-TPUS for the detection and classification of anal fistula.In this prospective study, 3D-TPUS reconstructions were performed before and after SVE enhancement in 60 patients with fistula-in-ano who intended to undergo surgery at the Department of Anorectal Surgery, Qilu Hospital, Cheeloo College of Medicine, Shandong University (P. R. China) between January 2021 and October 2021. Accuracies of anal fistula classification, complexity classification, detection of anal fistula branches, and detection of internal opening between 3D-TPUS and SVE 3D-TPUS were compared based on a reference standard-intraoperative findings.This study enrolled 60 patients (mean age, 37.1 ± 11.4 years; mean follow-up, 9 ± 3 months). Intraoperative findings showed that the fistula type was intersphincteric in 23 patients (38.3%), trans-sphincteric in 35 (58.3%; 12 high and 23 low), and suprasphincteric in 2 (3.3%). Moreover, 68 internal openings were found. Compared with the accuracy of 3D-TPUS, that of SVE 3D-TPUS was similar in fistula classification [95.0% (57/60) vs 96.7% (58/60), P = 0.392], but significantly higher in internal opening evaluation [80.9% (55/68) vs 97.1% (66/68), P = 0.001], complexity classification [85.0% (51/60) vs 98.3% (59/60), P = 0.018], and detection of fistula branches [70.4% (19/27) vs 92.6% (25/27), P = 0.031].SVE 3D-TPUS may be a useful examination for patients with perianal fistulae because of its high accuracy and consistency with intraoperative findings, especially in complex fistula-in-ano and difficult cases.© The Author(s) 2024. Published by Oxford University Press and Sixth Affiliated Hospital of Sun Yat-sen University.