Prostate cancer is prevalent among cancers in men. A simple method for screening of reliable biomarkers is pivotal for early detection of prostate cancer. Prostate-specific antigen (PSA) has been a commonly used biomarker for prostate cancer, in spite of its false-positive limitation. On the other hand, alpha-methylacyl-CoA racemase (AMACR), a metabolic enzyme, has been proven to be a highly expressed biomarker in prostate cancer cells. Therefore, a method or tool, which can detect either PSA or AMACR or both simply, cost effectively, and with high sensitivity and selectivity is desirable. We describe a novel bioconjugated, single-use biosensor capable of detecting both PSA and AMACR antigens in undiluted human serum. The preparation of the biosensor by the bioconjugation mechanism occurred within a day, which could be completed prior to actual testing. The effectiveness of the bioconjugation mechanism and the coverage of the electrode surface of the biosensor were experimentally assessed. Measurements of PSA and AMACR antigens and the specificity of the biosensor were carried out using differential pulse voltammetry. This biosensor was single-use and cost-effective and required a small quantity of test medium and relatively short preparation time, providing a very attractive biosensor for the detection of the biomarkers of prostate cancer.