The multilayers of polycation-based non-viral DNA nanoparticles and biodegradable poly(L-glutamic acid) (PGA) were constructed by a layer-by-layer (LbL) technique. Poly(ethyleneimine) (PEI) was used to condense DNA to develop non-viral DNA nanoparticles. AFM, UV-visible spectrometry, and TEM measurements revealed that the PEI-DNA nanoparticles were successfully incorporated into the multilayers. The well-structured, easily processed multi-layers with the non-viral DNA nanoparticles may provide a novel approach to precisely control the delivery of DNA, which may have great potential for gene therapy applications in tissue engineering, medical implants, etc.