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This study introduces a novel tyrosinase-based biosensor designed to simultaneously detect tyrosinase, tyrosine, L-DOPA, and L-DOPA quinone. The biosensor, designated as GCE/GNP/Cys/Chit/Tyrase, was developed by immobilizing tyrosinase on a modified glassy carbon electrode (GCE) that incorporates electrodeposited gold nanoparticles, cysteine, and chitosan. The morphology of the biosensor was characterized using scanning electron microscopy (SEM). The electrochemical behaviors of the biosensor were explored in response to the target analytes. Key analytical characteristics were assessed, including linear range, sensitivity, selectivity, limits of detection and quantification, long-term stability, repeatability, reproducibility, electrochemically active surface area, and charge transfer behavior. The biosensor demonstrated a linear response range of 1–120 μM, with an impressive sensitivity of 200.4 mA.Lmol−1cm−2 and a detection limit of 27 μM. This study provides a comprehensive evaluation of the analytical features of tyrosinase-based biosensors. Additionally, the biosensor was applied to quantify dopamine in brain tissue, utilizing a calibration curve derived from the fabricated biosensor. The dopamine concentration measured in five Wistar rats was 35 ± 2.75 μM, reflecting the mean and standard deviation, respectively. These results confirm the biosensor's capability for accurately detecting this critical neurotransmitter in the brains of Wistar rats. This investigation underscores the potential of tyrosinase-based biosensors for diverse analytical applications in biological samples.