Tyramine (TYM) serves as a critical indicator of microbial spoilage and significant health risks at elevated concentrations. Herein, we report an integrated experimental and computational approach to develop a high-performance electrochemical biosensor for selective TYM detection. Tyrosinase (Tyro) was immobilized onto carboxylated single-walled carbon nanotube-modified screen-printed carbon electrodes (Tyro-COOH-SWCNT/SPCE) to construct a nanostructured bioelectrode. The resulting biosensor exhibits a detection limit of 1.15 × 10-6 M and a linear range from 9.98 × 10-6 to 1.08 × 10-4 M. Furthermore, the biosensor demonstrates high selectivity toward TYM, with minimal interference (<10%) from common biogenic amines, including histamine, putrescine, spermidine, and spermine. Practical applicability was validated using spiked chicken meat samples, achieving satisfactory recoveries between 89.10-108.59%. To provide a molecular-level interpretation of TYM recognition by Tyro, molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area (MMPBSA) analysis was performed. The atomistic simulations of Tyro reveal a copper-stabilized binding pocket that accommodates TYM through predominantly hydrophobic interactions. Molecular mechanics Poisson-Boltzmann surface area (MMPBSA) analysis yields a favorable binding free energy (ΔG = -22.46 ± 2.26 kcal mol-1), while per-residue decomposition identifies key histidine and valine residues near the binuclear copper center as major contributors to binding. By integrating experimental electrochemical performance with atomistics insights into enzyme-substrate recognition, this study provides a mechanism-informed framework for understanding Tyro-based biosensing and demonstrates its potential for rapid, selective, and on-site monitoring of food spoilage.