The layer-by-layer (LbL) self-assembly of poly-l-lysine (PLL) and deoxyribonucleic acid (DNA) was used to construct the enzymatic biodegradable multilayered films. The LbL build up of DNA multilayers was monitored by UV-vis spectrometry, and atomic force microscopy (AFM). AFM, UV-vis spectrometry and fluorescence spectrometry measurements indicated that 90% of DNA within the films was released almost linearly under 5 U mL(-1)alpha-chymotrypsin in PBS at 37 degrees C in 35 h. TEM and zeta potential experiments revealed that the released DNA molecules were condensed into the slight positive complexes with size from 20 to several hundred nanometers. The well-structured, easy processed enzymatic biodegradable multilayered film may have great potential for gene applications in tissue engineering, medical implants, etc.