A dual-modality microfluidic biosensor is fabricated using a mesoporous nanostructured cysteine-graphene hydrogel for the quantification of human cardiac myoglobin (cMb). In this device, the nanoengineered mesoporous l-cysteine-graphene (Cys-RGO) hydrogel performs the role of a dual-modality sensing electrode for the measurements conducted using differential pulse voltammetry and surface plasmon resonance (SPR) techniques. High surface reactivity, mesoporous structure and fast electron transfer combined with good reaction kinetics of the graphene hydrogel in this device indicate excellent performance for the detection of human cardiac myoglobin in serum samples. In electrochemical modality, this microfluidic chip exhibits a high sensitivity of 196.66 μA ng-1 mL cm-2 for a linear range of concentrations (0.004-1000 ng mL-1) with a low limit of detection (LOD) of 4 pg mL-1 while the SPR technique shows a LOD of 10 pg mL-1 for cMb monitoring in the range 0.01-1000 ng mL-1. The intra-assay coefficient of variation was less than 8% for standard samples and 9% for real serum samples, respectively. This Cys-RGO hydrogel-based microfluidic SPR chip allows real-time dynamic tracking of cMb molecules with a high association constant of 4.93 ± 0.2 × 105 M-1 s-1 and a dissociation constant of 1.37 ± 0.08 × 10-4 s-1, self-verification, reduced false readout, and improved detection reliability.