We evaluated the effect of Tween 80 as elicitor on licochalcone A from hairy root cultures of Glycyrrhiza uralensis Fisch. After a 15-days treatment with 2% Tween 80, hairy roots still grew well and produced higher levels of licochalcone A and total flavonoids than the control (without treatment). Licochalcone A content and total flavonoid content were 3.103 and 127.095 mg per flask (9- and 11-fold higher), respectively, compared with controls. Secretion of licochalcone A and total flavonoids into the culture medium was remarkably high, up to 98 and 94% of the total production, respectively. The enhanced flavonoid production was associated with elevated mRNA levels and enzyme activities of phenylalanine ammonia-lyase (PAL), 4-coumarate:coenzyme A ligase (4CL), and cinnamate-4-hydroxylase (C4H). These results clearly demonstrated that Tween 80 treatment permeabilized the roots to enhance secretion, but also acted as an efficient elicitor of licochalcone A and total flavonoid production in hairy roots of G. uralensis Fisch.