Abstract: The effects of extracting temperature, extracting time, ethanol concentration and solid-liquid ratio on extraction yield of total flavonoids from glycyrrhiza residue were investigated by single factor experiments.Based on single factor experiments, optimum parameters for extraction were selected with a L9 (34) form by orthogonal experiment design. Results showed that the optimized extraction conditions were as follows :80% ( V /V) ethanol, 80 ℃, 1 ∶ 20 ( g /m L) solid-liquid ratio, and 105 min extraction time, under which the maximum extraction yield of total flavonoids was 37.387mg/g. Enzymatic pretreatment and microbial pretreatment of glycyrrhiza residue were further performed, respectively. Results indicated the extraction yield of total flavonoids was indeed enhanced by enzymatic pretreatments, among which the pretreatment with the combination of celluluase and xylanase gave the best effect. For biological pretreatments, the extraction yield of total flavonoids was the highest after pretreatment by Phanerochaete chrysosporium for 12 days, reaching 45.38mg/g. Moreover, comparison of all the pretreatments revealed that microbial pretreatment not only enhanced the extraction yield of total flavonoids significantly, but also decreased the ethanol concentration, extracting temperature and time.The research had significant reference value for comprehensive development and utilization of glycyrrhiza residue.