Pinocembrin is a flavonoid that exhibits diverse biological properties. Although the major source of pinocembrin is propolis, it can be synthesized biologically using microorganisms such as Escherichia coli, which has been used to synthesize diverse natural compounds. Pinocembrin is synthesized from phenylalanine by the action of three enzymes; phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), and chalcone synthase (CHS). In order to synthesize pinocembrin from glucose in Escherichia coli, the PAL, 4CL, and CHS genes from three different plants were introduced into an E. coli strain. Next, we tested the different constructs containing 4CL and CHS. In addition, the malonyl-CoA level was increased by overexpressing acetyl-CoA carboxylase. Through these strategies, a high production yield (97 mg/l) of pinocembrin was achieved.