Cancer stands as a prominent global cause of mortality, necessitating early detection to augment survival rates and alleviate economic burdens on healthcare systems. In particular, prostate cancer (PCa), impacting 1.41 million men globally in 2020, accentuates the demand for sensitive and costeffective detection methods beyond traditional prostate-specific antigen (PSA) testing. While clinical techniques exhibit limitations, biosensors emerge as compact, user-friendly alternatives to traditional laboratory approaches. However, existing biosensors predominantly concentrate on PSA detection, prompting the necessity for advancing toward multiplex sensing platforms. This study introduces a compact opto-microfluidic sensor featuring a substrate of gold nanospikes, fabricated via electrodeposition, for enhanced sensitivity. Embedded within a microfluidic chip, this nanomaterial enables the precise and concurrent measurement of PSA, alongside two complementary PCa biomarkers, matrix metalloproteinase-2 (MMP-2) and anti-α-methylacyl-CoA racemase (anti-AMACR) in diluted human plasma, offering a comprehensive approach to PSA analysis. Taking advantage of the localized surface plasmon resonance principle, this biosensor offers robustness and sensitivity in real sample analysis without the need for labeling agents. With the limit of detection at 0.22, 0.37, and 0.18 ng/mL for PSA, MMP-2, and anti-AMACR, respectively, this biosensing platform holds promise for point-of-care analysis, underscoring its potential impact on medical diagnostics.