We present a microfluidic assay device integrated with Au nanorods for rapid and sensitive detection of Cardiac troponin I (cTnI), the current gold standard biomarker for diagnosing acute myocardial infarction. The Au nanorods, fabricated using oblique angle deposition (OAD), significantly enhance fluorescent signals, thereby eliminating the need for high-resolution lithography technologies. Leveraging this device, rapid and sensitive detection of cTnI is realized with a limit of detection (LOD) of 6.9 pg mL−1 within 6 minutes, referring to a sensitivity enhancement of 4000-fold compared to glass substrates. Given its simple fabrication method, ease of operation, and highly sensitive immunoassay performance, this device holds great potential for clinical and point-of-care testing (POCT) applications.