Aim: The upgrading or staging in men with prostate cancer (PCA) undergoing active surveillance (AS), defined as Gleason score (GS) ≥ 3+4 or more than 2 area with cancer, was investigated in our experience using the software-based fusion biopsy (FB). Methods: We selected from our database, composed of 620 biopsies, only men on AS according to criteria of John Hopkins Protocol (T1c, < 3 positive cores, GS = 3+3 = 6). Monitoring consisted of PSA measurement every 3 months, a clinical examination every 6 months, confirmatory FB within 6 months and then annual FB in all men. The suspicious MRI lesions were scored according to the Prostate Imaging Reporting and Data System (PI-RADS) classification version 2. FB were performed with a transrectal elastic free-hand fusion platform. The overall and clinically significant cancer detection rate was reported. Secondary, the diagnostic role of systematic biopsies was evaluated. Results: We selected 56 patients on AS with mean age 67.4 years, mean PSA 6.7 ng/ml and at least one follow-up MRI-US fusion biopsy (10 had 2 or 3 follow-up biopsies). Lesions detected by MRI were: PIRADS-2 in 5, PIRADS-3 in 28, PIRADS-4 in 18 pts and PIRADS-5 in 5 patients. In each MRI lesion, FB with 2.1 ± 1.1 cores were taken with a mean total cores of 13 ± 2.4 including the systematic cores. The overall cancer detection rate was 71% (40/56): 62% (25/40) in target core and 28% (15/40) in systematic core. The overall significant cancer detection rate was 46% (26/56): 69% (18/26) in target vs 31% (8/26) in random cores. Conclusions: The incidence of clinical significant cancer was 46% in men starting active surveillance, but it was more than doubled using MRI/US Target Biopsy 69% (18/26) rather than random cores (31%, 8/26). However, 1/3 of disease upgrades would have been missed if only the targeted biopsies were performed. Based on our experience, MRI/US fusion target biopsy must be associated to systematic biopsies to improve detection of significant cancer, reducing the risks of misclassification.