Prostate cancer remains the second-most common cancer diagnosed in men, despite the increasingly widespread use of serum prostate-specific antigen (PSA) screening. The controversial clinical implications and cost benefits of PSA screening have been highlighted due to its poor specificity, resulting in a high rate of overdiagnosis and underdiagnosis. Thus, the development of novel biomarkers for prostate cancer detection remains an intriguing challenge. Urine is emerging as a source for prostate cancer biomarker discovery.Currently, new urine biomarkers already outperform serum PSA in clinical diagnosis. Meanwhile, the advances in nanotechnology have provided a suite of diagnostic tools to study prostate cancer in more detail, sparking a new era of biomarker discoveries. In this review, we envision that future prostate cancer diagnosis will probably integrate multiplex nano-diagnostic approaches to detect novel urinary biomarkers. However, challenges remain in differentiating indolent from aggressive cancers to better inform treatment decisions, and clinical translation still needs to be overcome.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.