We developed a new plasmonic nanostripe microcone array (PNMA) substrate-integrated microfluidic chip for the simultaneous surface-enhanced Raman scattering (SERS)-based immunoassay of the creatine kinase MB isoenzyme (CK-MB) and cardiac troponin (cTnI) cardiac markers. The conventional immunoassay usually employs a microtiter plate as the solid capture plate to form the immunocomplexes. However, the two-dimensional (2D) surface of the microtiter plate limits the capture efficiency of the target antigens due to the steric hindrance effect. To address this issue, a gold film-coated microcone array with nanostripes was developed that can provide a large surface area for capture antibody conjugation and serve as a SERS-active substrate. This unique nano-microhierarchical structure showed an excellent light trapping effect and induced surface plasmon resonance to further enhance the Raman signals of the SERS nanoprobes. It significantly improved the sensitivity and applicability of SERS-based immunoassay on the microfluidic chip. With this integrated microfluidic chip, we successfully performed the simultaneous detection of CK-MB and cTnI, and the detection limit can reach 0.01 ng mL. It is believed that the PNMA substrate-integrated microfluidic chip would play a critical role in the rapid and sensitive diagnostics of cardiac diseases.