Abstract: Objective To compare the clinical values of transperineal （TP） and transrectal （TR） ultrasound-guided prostate biopsy in the diagnosis of prostate cancer. Methods Clinical data of 558 patients undergoing ultrasound-guided prostate biopsy were collected. All patients were divided into the TP （n = 425） and TR groups （n = 133）. The biopsy outcomes were recorded in two groups. The PSA level and positive rate were statistically compared between two groups. The positive rate in patients with PSA levels of < 10 ng/ml, 10-30 ng/ml and > 30 ng/ml was compared between two biopsy methods. The incidence of complications was observed in two groups. Results The positive rate of prostate cancer was 40% （170/425） in the TP group and 44.4% （59/133） in the TR group with no significant difference （P > 0.05）. In the TP group, the positive rate of prostate cancer in patients with PSA levels of < 10 ng/ml, 10-30 ng/ml and > 30 ng/ml was 22%, 26% and 83%, which did not significantly differ from 24%, 24% and 84% in the TR group （all P > 0.05）. The incidence of hematuria, acute urinary retention and fever did not significantly differ between two groups （all P > 0.05）, whereas the risk of rectal bleeding through transrectal route was remarkably higher than that of the perineal route （P < 0.001）. Conclusion Both TP and TR prostate biopsy methods are safe and efficacious, and TP approach can lower the risk of rectal bleeding.