In this study, atomic force microscopy (AFM) was employed to characterize the microstructure of chitosan-zinc oxide (ZnO) nanocomposite. Three dimensional image of AFM indicated that ZnO nanoparticles were in the ranging of 0.25-33.33 nm in height, 13-177 nm in diameter (confirmed by particle size analyzer), irregular and triangular cluster in morphologies. Furthermore, ZnO nanoparticles were well incorporated into chitosan solution indicated by UV-vis absorption peak of 359 and 341 nm for ZnO alone and nanocomposite respectively. AFM revealed the relatively continuous matrix without pores, smooth and contoured film were formed from chitosan alone as well as chitosan-ZnO composites. There were no remarkable different for surface roughness of both films indicating ZnO in nano-scale were blended well with chitosan matrix.