Prostate-specific antigen (PSA) screening lacks specificity, particularly within the 4–10 ng/mL diagnostic gray zone, leading to unnecessary prostate biopsies and overtreatment. Here, we developed and validated a streamlined urine DNA methylation assay for non-invasive prostate cancer (PCa) detection and biopsy decision-making. Tissue-based Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) was integrated with large-scale public methylome cohorts to identify PCa-specific hypermethylated regions. Candidate markers were further refined by targeted bisulfite sequencing (TBS) and translated into quantitative PCR (qPCR) assays using post-prostatic massage urine samples. In a retrospective development cohort of 348 participants, stepwise marker selection and logistic regression modelling generated a compact two-gene methylation assay comprising ADD3_2 and GSX2_2. The model was then evaluated in an independent prospective multicenter cohort of 200 participants. The assay achieved robust diagnostic performance, with areas under curve (AUC) of 0.86 in the development cohort and 0.91 in the validation cohort, while maintaining specificity above 90%. Performance was preserved in the PSA gray zone and for clinically significant PCa (csPCa). Decision curve analysis (DCA) indicated that integrating this tool could safely avert approximately 55% of unnecessary biopsies without compromising cancer detection. This simple qPCR-based two-gene urine methylation assay provides a scalable, cost-effective and non-invasive complement to PSA-based PCa screening.