2,3,5,6-Tetramethylpyrazine (TMP), an organic compound with numerous potential health benefits, is extensively used in medical and food products. This research introduces a novel approach for the precise quantification of TMP by employing a poly(L-lysine) modified glassy carbon electrode (pLL/GCE). The pLL/GCE is fabricated through the electrochemical polymerization of L-lysine onto a glassy carbon electrode (GCE) surface. This modification enhances the electrode's electrocatalytic efficiency, resulting in a significant increase in the reduction peak current of TMP. Exploiting this distinctive property, we establish an electrochemical method for quantifying TMP using cyclic voltammetry (CV). Optimized conditions reveal a robust linear correlation between the reduction peak current of TMP and its concentration within two adjacent linearity ranges: 40.0 nmol/L to 1.0 μmol/L and 1.0 μmol/L to 100.0 μmol/L. Notably, this method achieves an impressive detection limit of 20.0 nmol/L. To validate the practicality of this approach, we analyzed various real-world samples, obtaining recovery rates ranging from 98.2% to 100.8%. The developed method holds significant promise for the determination of TMP in medical and food products, underscoring its potential for widespread application.