Abstract: In this study, lactose was used as raw material, on the basis of single factor experiment and combined with response surface methodology, enzyme addition, reaction temperature, reaction time, reaction pH and other factors were utilized to investigate the influence of the galactooligosaccharides total yield and the galactotetraose yield. The process of enzymatic preparation of galactooligosaccharides by β-galactosidase was optimized. The results showed that the optimum process parameters for the enzymatic preparation of galactooligosaccharides were as follows: The initial lactose concentration was 300 g/L, the amount of enzyme added was 8.25 U/g lactose, the reaction temperature was 49 ℃, the reaction time was 16 h, and the reaction pH was 5.6. Under these conditions, the galactooligosaccharides total yield was 14.61% and the galactotetraose yield was 3.31%. The method selectively improved the yield of galactooligosaccharides with high degree of polymerization, which would provide a certain theoretical reference for the functional application of galactooligosaccharides and the further study and development of special medical food.