The fabrication of 2D materials and polymer-based nanocomposites deposited on flexible conductive interfaces has unblocked new horizons to expedite reaction kinetics for developing highly selective and sensitive electrochemical biosensors. Herein, we developed a novel biosensing platform, comprising graphene oxide and a silk fibroin-based nanocomposite, drop-cast on a carbon cloth electrode. The fabricated interface was expected to be a robust and miniaturized sensing platform for precise detection of dopamine (DA). Characterization was performed by SEM, EDX, FTIR, XRD, UV-visible spectroscopy, contact angle measurement, fluorescence spectroscopy, particle size, and zeta potential analysis. CV, EIS, DPV, and chronoamperometry demonstrated the superior electrochemical properties of the working interface and revealed its enhanced active surface area, increased conductivity, and accelerated electron transfer rate. The designed interface exhibited low LoD (0.41 μM), admirable stability, good sensitivity (2.46 μA μM⁻¹ cm⁻²), wide linearity ranging from 100–900 μM, excellent reproducibility, and superb selectivity against dopamine even in the presence of possible interfering analytes. These findings endorse the feasibility of the practical execution of such an integrated system in real sample analysis.