Abstract: In this study,total flavonoids from spinach were extracted by cellulase combined with ultrasonic extraction.The effects of methanol concentration,enzyme addition,material ratio,extraction temperature,enzymatic hydrolysis pH and extraction time on the yield of total flavonoids were investigated by single factor experiment,the extraction conditions were optimized by orthogonal test. Results showed that the optimum conditions of extraction for total flavonoids of spinach were as follows:With 70% methanol solution as extractant and 1.2% enzyme addtion,material liquid ratio 1:30 g/mL,extraction temperature 60℃,enzymolysis pH5.0 and extraction time 45 min,flavonoids in this condition could reach a yield of 15.56%. Seven flavonoid compounds,gallic acid,chlorogenic acid,catechin,rutin,apigenin,kaempferol and quercetin,were separated by high performance liquid chromatography(HPLC). The measured contents were 0.83,1.37,0.64,1.46,2.12,1.25,1.87 mg/g,respectively. The results showed that the linear relationship of flavonoid monomers was good,the recovery accuracy was high and the relative standard deviation was good,which could be used for the qualitative and quantitative determination of flavonoids in spinach.