This study aimed to evaluate the learning curve of transperineal magnetic resonance imaging (MRI)/ultrasound (US) fusion biopsy in a team composed of a single surgeon, a single radiologist, and a single pathologist. We prospectively enrolled 206 patients undergoing MRI/US fusion prostate biopsy and divided them into four cohorts by the year of biopsy. We analyzed temporal changes in clinically significant prostate cancer (csPC) detection rate, percentage of positive cores on biopsy, and Gleason upgrading rate after radical prostatectomy. The csPC detection rate by MRI/US fusion targeted biopsy (TB) increased significantly (from 35.3% to 60.0%, p = 0.01). With increased experience, the csPC detection rates for small ($\le$1 cm) and anterior target lesions gradually increased (from 41.2% to 51.6%, p = 0.5; from 54.5% to 88.2%, p = 0.8, respectively). The percentage of positive cores on TB increased significantly (from 18.4% to 44.2%, p = 0.001). The Gleason upgrading rate gradually decreased (from 22.2% to 11.1%, p = 0.4). In conclusion, with accumulated experience and teamwork, the csPC detection rate by TB significantly increased. Multidisciplinary team meetings and a free-hand biopsy technique were the key factors for overcoming the learning curve.