Development of materials with stimuli-responsive properties is of interest for biotechnical applications including gene delivery and regenerative medicine. Here, we report a multilayer film through layer-by-layer self-assembly of DNA polyplexes and charge-reversible poly(ethylenimine) (cPEI). Through functionalizing PEI with cyclohexanedicarboxylic acid, cPEI showed negatively charged and therefore was used for electrostatic self-assembly with positively charged DNA polyplexes. Side chains of cPEI can be hydrolyzed in acidic environment while it is stable in neutral condition. Such pH-triggered hydrolysis led to charge reverse of cPEI from negative to positive, which consequently led to a disassembly of multilayer film. Both UV-vis and ellipsometry spectrum measurements suggested that the multilayer film grew with a thickness of 150 nm for twelve bilayers. Under low pH condition, the multilayer film collapsed and DNA polyplexes were released. The multilayer film containing cPEI could be served as a local gene delivery system in specific low pH conditions such as extracellular acidity of solid tumor and lysosomal. (C) 2012 Elsevier B.V. All rights reserved.