The effects of ultrasonic, microwave and their synergistic action on the release of free amino acid, 5′-nucleotides, organic acid and soluble sugar of flammulina velutipes were studied. The results showed that the total amount of flavor substances from the extracting solution using these three methods are higher than hot water extraction. Comparing the three methods, the content of free amino acid, 5′-nucleotides, organic acid and soluble sugar using ultrasonic-microwave synergistic action is higher than microwave and ultrasonic. The ultrasonic-microwave is synergistic method is more effective on releasing flavor substances from flammulina velutipes.