The use of emerging nanotechnologies, such as plasmonic nanoparticles in diagnostic applications, potentially offers opportunities to revolutionize disease management and patient healthcare. Despite worldwide research efforts in this area, there is still a dearth of nanodiagnostics which have been successfully translated for real-world patient usage due to the predominant sole focus on assay analytical performance and lack of detailed investigations into clinical performance in human samples. In a bid to address this pressing need, we herein describe a comprehensive clinical verification of a prospective label-free surface-enhanced Raman scattering (SERS) nanodiagnostic assay for prostate cancer (PCa) risk stratification. This contribution depicts a roadmap of (1) designing a SERS assay for robust and accurate detection of clinically validated PCa RNA targets; (2) employing a relevant and proven PCa clinical biomarker model to test our nanodiagnostic assay; and (3) investigating the clinical performance on independent training (n = 80) and validation (n = 40) cohorts of PCa human patient samples. By relating the detection outcomes to gold-standard patient biopsy findings, we established a PCa risk scoring system which exhibited a clinical sensitivity and specificity of 0.87 and 0.90, respectively [area-under-curve of 0.84 (95% confidence interval: 0.81–0.87) for differentiating high- and low-risk PCa] in the validation cohort. We envision that our SERS nanodiagnostic design and clinical verification approach may aid in the individualized prediction of PCa presence and risk stratification and may overall serve as an archetypical strategy to encourage comprehensive clinical evaluation of nanodiagnostic innovations.