This experiment was conducted to study the effects of different factors on protein extraction rate of high-temperature defatted soybean meal. Using the high-temperature defatted soybean meal as a raw material, soy protein was extracted by alkali-solution and acid-isolation assisted by ultrasound, and single factor and orthogonal experiments were performed to investigate the effects of extractive temperature, water to flour, pH, extractive time, crushing fineness, ultrasonic time and ultrasonic power on the extraction rate of soybean protein. The results showed as follows: 1) the condition which could significantly increase the protein extraction rate was: crushing fineness ≥ 40 mesh, pH ≥9; extractive temperature ≥ 30 ℃;water to flour ≥20: 1 (mL/g); ultrasonic power 300 to 400 W, ultrasonic time ≥5 min. 2) The factor which could affect the rate of protein extraction was: extractive time > ultrasonic power > water to flour > ultrasonic time > crushing fineness > extractive temperature > pH. It is concluded that within limits, with the increase of factor level, the protein extraction rate is improved. the optimal combination of Extraction conditions is: extractive time 45 min, ultrasonic power 500 W, water to flour 25: 1 (mL/g), ultrasonic time 15 min, crushing fineness 60 mesh, extractive temperature 45 ℃ and pH 9.0.