Buffalo milk safety was highlighted with the increase in dietary consumption, and a little information is available on buffalo milk allergy except for cross-reactivity between buffalo and cow milk. In this work, linear epitopes and critical amino acids of buffalo β-lactoglobulin were defined by 4 rabbit’s sera using SPOTTM peptide arrays approach based on the defined mimotopes. The eight epitopes on buffalo β-lactoglobulin were located in the position of A6(21-30), A7(25-34), A8 (29-38), B4 (73-82), B5(77-86), C(87-96), F4(134-143) and F8(150-159), respectively. Among them, four epitopes (A7, A8, F4 and F8) were described as the most major epitopes and peptide (A6, B4, B5 and C) as the second major epitopes. Following single AA substitutions (Alanine or Glycine) at each position of the major epitopes, 2,3,2,3,5 and 3 of critical amino acids were identified on epitopes of A6, A8, B5, C , F4 and F8, respectively, which vary in distribution among the epitopes, such as in C terminal or N terminal and in continuous or discontinuous forms, characteristics including hydrophobicity, polar and charge, and existed frequency.