The interaction of tyrosinase with sulfonated starch-graft-polyaniline@graphene (SSt-g-PANI@G) nanocomposite was investigated by electrochemical methods. The activity of the immobilized tyrosinase (Tyase) was proved by the electrochemical detection of three substrates (L-dopa, caffeic acid, and catechol). The SSt-g-PANI@G nanocomposite was characterized by Fourier-transform infrared spectra (FT-IR), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), energy-dispersive X-ray analysis (EDX), and thermogravimetric analysis (TGA). To immobilize tyrosinase on the surface of the nanocomposite, a simple drop-casting technique was used. The presence of sulfuric acid and hydroxyl groups in SSt, amine groups in PANI, and high surface-to-volume ratio and electrical conductivity of graphene in the prepared nanocomposite led to good enzyme immobilization on the electrode surface. The modified electrode showed a suitable catalytic effect on the electrochemical redox agent, compared with the bare electrode. The peak current responses for three substrates were studied with a calibration curve derived using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). In addition, the fabricated SSt-g-PANI@G/Tyase/GCE showed a more suitable response to catechol, L-dopa, and caffeic acid substrates, respectively.