Objective To investigate the relationship between low testosterone levels and prostate cancer detection risk in a biopsy population. Patients and Methods In all, 681 men who underwent initial 12-core transrectal prostate biopsy at our institution were included in this retrospective study. Patients were divided into groups with low (= 300 ng/dL). Clinical and pathological data were analysed. Results Among 681 men, 86 men (12.6%) had low testosterone levels, 143 (32.7%) had a positive biopsy, and 99 (14.5%) had high-grade prostate cancer. The mean age, prostate-specific antigen (PSA) level, PSA density, body mass index (BMI), number of abnormal digital rectal examination (DRE) findings, and diabetes mellitus (DM) history were significantly different between the low and normal level testosterone groups. A low testosterone level was significantly associated with a higher risk of detection of overall prostate cancer than a normal testosterone level in univariate analysis (odds ratio [OR] 2.545, P = 0.001), but not in multivariate analysis adjusting for parameters such as age, PSA, prostate volume, BMI, abnormal DRE findings and DM (OR 1.583, P = 0.277). Meanwhile, a low testosterone level was significantly related to a higher rate of high-grade prostate cancer compared with a normal testosterone level in univariate (OR 3.324, P < 0.001) and multivariate analysis adjusting for other parameters (OR 2.138, P = 0.035). Conclusion Low testosterone level is an independent risk factor for high-grade prostate cancer detection at biopsy. Therefore, checking testosterone levels could help to determine whether prostate biopsy should be carried out.