The optimal conditions of total flavonoids extraction from Mentha spicata were identified by orthogonal test. The results showed that the ethanol concentration and the ultrasonic time had significant effect on the extraction of total flavonoids and the optimum conditions of extraction were A3B3C1D1, which were as follows: the ethanol concentration was 50%, the ultrasonic time was 40 min, the solid-liquid ratio was 1 g: 50 mL and the temperature of extraction was 60 degrees C, On the basis of the extracting method above, the content of total flavonoids in Mentha spicata was 0.0951 mg/mg. The linear equation was A = 11.51143C - 0.00029, r = 0.9999. This method is simple, quick and accurate with good prosperity.