The coacervate formation and interaction between α-lactalbumin (ALA) and gum arabic (GA) was investigated with turbidimetric analysis, fluorescence spectroscopy, atom force microscopy (AFM), and isothermal titration calorimetry (ITC). Critical pH values (pHc, pHφ1, pH opt and pHφ2) of phase transitions for ALA-GA complexes (1:1, 0.1%, w/v) without NaCl, representing the formation of soluble and insoluble complexes, were 4.95, 3.85, 3.70, and 2.30, respectively. pH and NaCl concentrations are dominant in influencing the formation of ALA-GA complexes. AFM graphs evidently indicated the formation, association, and dissociation of ALA-GA complex. ITC results confirmed that the formation of soluble ALA/GA complex was a spontaneous exothermic process, and the electrostatic interaction between ALA and GA was stronger at pH 3.0 than that at pH 4.9 and 5.6. The obtained information of this study provided insights into the interaction between ALA and GA and expand the application of ALA-based formulation in food system with desirable functionality.