Aim: Prostate cancer (PCa) is the most commonly diagnosed non-skin cancer among men. Serum prostate-specific antigen level is used as a standard PCa biomarker for over 20 years. However, it has only 33％ specificity and 86％ sensitivity (for the cutoff value for prostate biopsy of > 4 ng/mL). This leads to overdiagnosis and overtreatment. In-depth insight into PCa metabolomics enables discovery of novel PCa biomarkers. Methods: Metabolomic altation in PCa serum, urine and interstitial fluid was examined using gold-nanoparticle-based laser mass spectrometry imaging. This study included 5 patients who underwent prostate biopsy with positive result, 5 patients with negative result and 10 healthy controls. Results: Over two hundred differentiating metabolites (87 in urine, 54 in serum and 78 in interstitial fluid) were detected. Four, twenty two and ten metabolites from urine, serum and interstitial fluid respectively showed statistical significant differential abundance between cancer and control group. Conclusion: Comprehensive metabolomic profile of PCa has been identified. Out of 36 metabolites, 20 were identified and should be further evaluated in clinical trials as a potential PCa biomarker. Urine concentration of triglyceride (12:0/20:1) showed over 10 times higher abundance in PCa samples in comparison to healthy controls and is considered the most promising potential biomarker.