BACKGROUND We investigated the feasibility of applying magnetic resonance imaging (MRI)-targeted biopsy (TB) in patients with prostate-specific antigen (PSA) levels <20 ng/mL. MATERIAL AND METHODS We retrospectively analyzed 218 patients with PSA levels <20 ng/mL and suspicious lesions according to the Prostate Imaging Recording and Data System version 2.0 (PI-RADS v2). All 218 men underwent transperineal MRI-TB, followed by template-guided 12-core systematic biopsy (SB). Of the 218 patients undergoing TB, 100 received MRI-ultrasound-assisted software fusion biopsy (FB) and 118 received cognitive biopsy (CB). Clinically significant prostate cancer (csPCa) was defined as a Gleason score ≥3+4. RESULTS The overall TB positive rate was similar to that of SB (P=0.156), but with a higher diagnostic rate for csPCa (P=0.034). SB misdiagnosed csPCa in 11.47% of cases; TB misdiagnosed csPCa in 5.50% of cases. SB+TB detected more tumors with a Gleason score of 7 than did SB alone (43 vs 22). Detection rates of csPCa were similar for CB and FB (P=0.217). In total, 47 men had 2 MRI-determined suspicious areas. Of 265 suspicious areas, 143 (53.96%) had a PI-RADS v2 score of 3; 92 (34.71%) had a score of 4; and 30 (11.32%) had a score of 5. The positive detection rates for csPCa in patients with PI-RADS v2 scores of 3, 4, and 5, were 11.19%, 48.91%, and 80.00%, respectively. CONCLUSIONS TB increased the positive biopsy detection rate but missed some cases of csPCa. TB combined with SB may be the most suitable biopsy for patients with PSA <20 ng/mL.