Background: Prostate biopsy is a crucial diagnostic tool for detecting clinically significant prostate cancer (csPCa). Traditional transrectal ultrasound (TRUS)-guided biopsy methods are often associated with an increased infection risk of infection and limited accuracy, particularly when diagnosing anterior lesions of the prostate gland. Objective: This article presented a structured protocol for performing transperineal fusion magnetic resonance imaging/ultrasound (MRI/US) prostate biopsy, highlighting its advantages over the TRUS approach. Our study included biopsy-naïve patients with elevated prostate-specific antigen levels or abnormal digital rectal examination findings, all of whom underwent pre-biopsy multiparametric MRI to guide targeted biopsies. The key objectives of this protocol were to improve the detection rates of csPCa, minimize infection risk, and standardize a transperineal technique that combines both systematic and targeted biopsies. In addition, we provided details on patient preparation, equipment requirements, procedural steps, and follow-up protocols to ensure the safety and effectiveness of the procedure. This protocol aims to serve as a guideline for institutions to adopt MRI/US fusion-guided transperineal biopsy, thereby enhancing diagnostic accuracy and patient safety. Conclusion: The transperineal fusion MRI/US biopsy protocol enhances diagnostic accuracy, particularly for anterior lesions, while reducing infections risks. Combining targeted and systematic biopsies improves detection rates of csPCa and offers a standardized, safe approach for clinical implementation.