In recent years, with the westernization of lifestyle, reduced physical activity and increased prostate-specific antigen (PSA) testing, the incidence of prostate cancer (PCa) has risen significantly in developing countries. Currently, PSA is the only PCa biomarker applied clinically, but it does not perform well in the early diagnosis and distinguishing between benign prostatic hyperplasia and prostate cancer. With the advances in deep sequencing technology, a series of new PCa biomarkers have been recently proposed to improve the diagnostic value of PSA, such as prostate cancer antigen 3 (PCA3), TMPRSS2-ETS fusion gene, microRNA, and other regulatory non-coding RNAs. In addition, the prostate health index (PHI) has been approved by the U.S. Food and Drug Administration (FDA) for clinical use in the detection of PCa. The prostate-specific membrane antigen (PSMA) has been confirmed to be specifically expressed on the surface of PCa cells. In this review, we provide an updated summary of the value and features of these novel biomarkers in the diagnosis and treatment of PCa.