In view of the difficult extraction and low protein content of millet isolated protein（MPI）， this experiment compared the extraction effect of MPI through alkali method and five kinds of enzymatic methods. Under the condition of combing α-amylase / glucoamylase / viscozyme L， the extraction yield and protein content of MPI were high. The molecular weight distribution and secondary structure information of MPI were not affected by different enzymatic methods except alkali method. Based on the results of 6 single-factor experiments， pH， temperature， enzyme addition amount and enzymatic hydrolysis time were selected as the main factors affecting the MPI protein content. Box-Behnken response surface analysis was conducted with the MPI protein content as the response value， and a mathematical regression model was established. Optimum conditions of tri-enzymatic digestion for extracting high protein content MPI were： pH 4.7， enzymatic hydrolysis temperature 48 ℃， enzymatic addition amount 2.2% and enzymatic hydrolysis time 10 h， under which protein content of MPI was （69.49±0.23）%。