Abstract:The extraction of total flavonoids from mulberry leaves was optimized by orthogonal array design with the objective of developing a sample preparation method for the spectrophotometric determination of total flavonoids with rutin as a standard reference material. Four extraction parameters were ranked in decreasing or－ der of importance as follows： material-liquid ratio， time， temperature and ethanol concentration. And optimum condition was confirmed by the orthogonal test. The best extraction process for extraction of mulberry leaves flavonoids was temperature 60 ℃， solid-liquid ratio 1 and 14 g/mL， 70 % ethanol concentration， extraction time 90 min， extracting 2 times. Under the best condition， the content of flavonoids was 50.2 mg/g.