Layer-by-layer (LBL) multilayer films were fabricated from poly(vinylalcohol) (PVA) and poly[acrylamide-co-3-(acrylamido) phenylboronic acid] [P(AAm-AAPBA)] by the use of covalent phenylboronate ester bonding as the driving force. The film thickness increases with decreasing pH and increasing ionic strength of the assembly solutions. Phenylboronate ester bonds in the film were confirmed from the IR marker mode at 1730 cm(-1). Because the phenylboronate ester bonding is reversible, the PVA/P(AAm-AAPBA) films disassemble gradually when immersed in aqueous solutions. The disassembly rate increases with increasing pH and decreasing ionic strength of the aqueous solutions. Furthermore, the disassembly of the film is accelerated by the addition of glucose, which competes with PVA for phenylboronic acid groups and weakens the interaction between the two polymers. Interestingly, the PVA/P(AAm-AAPBA) film presents glucose-sensitive behavior even under physiological conditions. The enhanced glucose-sensitive behavior at physiological pH may originate from the stabilization of phenylboronate ester by the adjacent amide group. These glucose-sensitive LBL films hold promise for self-regulated insulin release.