The optimum conditions of extraction of flavonoids from Pleurotus eryngii were studied by ultrasonic assisted complex enzyme method. Box-Behnken test of three factors and three levels was performed based on single factor test. Other factors fixed, the ratio of solid to liquid, ultrasonic time and enzymolysis pH were selected. Response surface analysis was performed by Design-expert 8.0.6. The results showed that the highest extraction rate was 3.352%, the ratio of material to liquid was 1:50（g/m L）, the ultrasonic time was 6 min and the pH value was 5.4.