BACKGROUND: Targeted and systematic transperineal biopsy of lesions guided by magnetic resonance imaging (MRI) and transrectal ultrasonography (TRUS) fusion technique may optimize the biopsy procedure and enhance the detection of prostate cancer. We described the transperineal biopsy guided by an automatic MRI-TRUS fusion technique, and evaluated the accuracy and feasibility of this method in a prospective single-center study. METHODS: The proposed method focuses on automating the delineation of prostate contours in both the MRI and TRUS images, the registration and fusion of MRI and TRUS images, the generation and visualiztion of the systematic biopsy cores in their corresponding locations within the 2D and the 3D views, as well as the computation and visualiztion of needle trajectories from preoperative planning to intraoperative navigation. A total of 76 patients with clinically suspected prostate cancer underwent systematic (SBx) and targeted (TBx) biopsies, all performed by a single urologist with more than 10 years of experience. The detection rates of prostate cancer (PCa) and clinically significant prostate cancer (csPCa) were recorded. We also measured preoperative registration time, duration of the overall surgical procedure, and postoperative complication rates within the first week following the surgery. Descriptive analyses were presented in this study. RESULTS: PCa was identified in 73.7% (56/76) of the subjects, while csPCa was identified in 61.8% (47/76). The preoperative registration time was 5.0 min (IQR: 4.4-6.0), while the overall surgery duration was 24.8 min (IQR: 23.2-27.2). Postoperatively, 12 patients experienced immediate hematuria, and one patient reported dysuria 1 day following surgery. CONCLUSIONS: The automatic MRI-TRUS fusion technique for transperineal biopsy is feasible and safe, with preoperative planning to intraoperative navigation it offering convenient and efficient preoperative preparation and surgical procedure.