Abstract: In this paper, peanut protein was extracted from peanut meal by alkaline solution and acid precipitation. The content of peanut protein was 87.7% by Kjeldahl method. Peanut polypeptides were prepared by ultrasonic-assisted enzymatic method. The effects of enzyme type, ultrasonic power, ultrasonic time, substrate concentration, enzyme addition amount and enzymolysis time on α-amylase inhibitory peptide were investigated using the yield of polypeptide and α-amylase inhibition rate as indexes. The optimal protease was flavor protease. Response surface test was designed to optimize the enzymolysis conditions on the basis of single factor. The optimal conditions were determined as follows: Ultrasonic power 150 W, ultrasonic time 30 min, liquid-solid ratio 20:1 mL/g, enzyme dosage 5000 U/g, enzymolysis time 2 h. Under these conditions, the yield of polypeptide was 41.92%, and the inhibitory rate of α-amylase was 50.62%, which was significantly higher than that of peanut polypeptide α-amylase which was 35.45% without ultrasonic assistance. According to the optimal process of α-amylase inhibitory peptide, the hydrolysate was separated by ultrafiltration and obtained four different fractions >10 kDa, 5~10 kDa, 3~5 kDa and <3 kDa, among which the molecular weight inhibition rate of <3 kDa fraction reached the highest 60.21%. Sephadex G-15 gel was used to separate and purify the <3 kDa components, and three components, P_1, P₂ and P₃ were obtained. The highest inhibition rate of P₃ reached 73.30%. Peanut peptide had good α-amylase inhibitory effect and had great application value in developing functional food.