A recent study showed that sarcosine may be potentially useful for the diagnosis and prognosis of prostate cancer (PCa). The aim of this study was to validate diagnostic value of sarcosine for PCa, to evaluate urine metabolomic profiles in patients with PCa in comparison of non-cancerous control, and to further explore the other potential metabolic biomarkers for PCa. Isotope dilution gas chromatography/mass spectrometry (ID GC/MS) metabolomic approach was applied to evaluate sarcosine using [methyl-D(3)]-sarcosine as an internal standard. Microwave-assisted derivatization (MAD) together with GC/MS was utilized to obtain the urinary metabolomic information in 20 PCa patients compared with eight patients with benign prostate hypertrophy and 20 healthy men. Acquired metabolomic data were analyzed using a two-sample t test. Diagnostic models for PCa were constructed using principal component analysis and were assessed with receiver-operating characteristic curves. Results showed that the urinary sarcosine level has no statistical difference between the PCa group and the control group. In addition, nine metabolomic markers between the PCa group and the healthy male group were selected, which constructed a diagnostic model with a high area under the curve value of 0.9425. We conclude that although urinary sarcosine value has limited potential in the diagnostic algorithm of PCa, urinary metabolomic panel based on GC/MS assay following MAD may potentially become a diagnostic tool for PCa.