L-alanine and β-alanine are important biological molecules and have zwitterion structure. In this study, L- alanine and β-alanine’s microscopic protonation constants and thermodynamic parameters (enthalpy, entropy, and free energy changes) for the proton–ligand systems of L-alanine methyl ester, β-alanine and β-alanine methyl ester have been determined at 5; 20; 35 °C, in ACN-water (25% ACN and 50% ACN (v/v)) mixtures at constant ionic strength of 0.1000 mol L–1 NaClO4 by potentiometric method. The results shown that, L-alanine and β-alanine’s microscopic protonation constants generally tend to decrease with temperature rise and their protonation reactions in ACN-water mixtures generally favor enthalpy-driven.