Rutin (RU) is a naturally occurring polyphenolic flavonoid with significant antioxidant properties found in various plants and natural sources. Due to its natural antioxidant activity, RU strengthens the blood vessels and inhibits harmful cancer cells. The current study involves the development of a highly sensitive electrochemical biosensor for detecting RU by modifying a glassy carbon electrode (GCE) using multi-walled carbon nanotubes (MWCNTs) and immobilization of the tyrosinase enzyme (Ty) via drop-casting onto the GCE surface. The evaluation of biosensor performance was illustrated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and Tafel plot studies. The electrochemical redox behavior of RU was thoroughly investigated, and a possible redox mechanism was proposed. The effect of supporting electrolyte phosphate buffer solution pH on RU's electrochemical behavior was studied, and pH 6.0 was identified as the optimal pH for further studies. The kinetic studies revealed that the detection process was adsorption controlled. From the scan rate experiments, the surface coverage concentration (Γ), heterogeneous rate constant (ks), and charge transfer coefficient (α) values were calculated. The biosensor showed impressive analytical performance, with a limit of detection of 4.47×10⁻⁸ M and a limit of quantification of 1.55×10⁻⁷ M, as found from the linear regression equation. The practical application of the biosensor was proved through tests for repeatability, reproducibility, and stability, confirming its potential for reliable and robust detection of RU.