The reactivity of amino acid residues in proteins towards quinones, along with the concentration of quinones, may play a crucial role in determining the specific sites of reaction between quinones and proteins. The reaction efficiencies of 20 different α-carbon amine group blocked amino acids with 4-methylbenzoquinone (4MBQ) were investigated at pH 7.0 by cyclic voltammetry. Among these, Nα-acetyl-L-lysine, Nα-Boc-L-histidine, Nα-acetyl-L-arginine, and Nα-acetyl-L-cysteine exhibited reactivity towards 4MBQ, with the reactivity sequence being: Nα-acetyl-L-cysteine > Nα-acetyl-L-lysine > Nα-acetyl-L-arginine > Nα-Boc-L-histidine. In addition, the reactivity of Nα-acetyl-L-cysteine towards 4MBQ was over 50-fold greater than that of Nα-acetyl-L-lysine, Nα-Boc-L-histidine and Nα-acetyl-L-arginine. Adducts formed from the reaction of 4MBQ with Nα-acetyl-L-lysine and Nα-acetyl-L-arginine were identified as quinone adducts. In contrast, those formed with Nα-acetyl-L-cysteine and Nα-Boc-L-histidine were identified as phenol adducts through UPLC-QTOF-MS/MS analysis. Irrespective of whether the molar ratio of 4MBQ to protein free thiol groups was 1:2 or 2:1, the residues of cysteine, lysine, arginine and histidine in β-lactoglobulin (β-Lg) invariably engaged in the reaction with 4MBQ. This work may provide valuable information for design polyphenol-protein covalent conjugates which can be further applied as functional additives in food industry by choosing protein with different proportion of lysine, histidine, arginine and cysteine residues.