In this work, to eliminate the effect of the hydrophobicity of N-acetyl groups in chitosan on the interaction between chitosan and DNA, a water soluble chitosan with molecular weight of 5000 and deacetylated degree of 99% was selected to complex with DNA at varied charged ratios. The physicochemical properties of chitoplexes were investigated by means of FTIR, circular dichroism (CD), static fluorescence spectroscopy, and atomic force microscopy (AFM). The results indicated that upon interacting with chitosan, the DNA molecules saved a B conformation, and the binding affinity of chitosan to DNA was dependent on pH of media. At pH 5.5, highly charged chitosan had a strong binding affinity with DNA; whereas in pH 12.0 medium, only weak interactions existed. The CD spectra of Hoechst 33258 competitive displacement revealed that chitosan was partially bound to the minor groove of DNA. The morphology of chitosan/DNA complexes was strongly dependent upon the charge ratios. At charge ratio (+/-) of 1:4, not all DNA could be entrapped in the complex; at ratio of 8:1, the spherical complexes with mean size of nanoscale were formed without free DNA, but no typical toroid patterns were observed, which might stem from the strong compact of DNA caused by highly charged chitosan. It was supposed that the strong interaction of chitosan with DNA possibly prevented gene unpacking from chitosan vector, consequently restraining gene expression in nucleus.