Fabrication of polymeric multilayered films based on the electrostatic self-assembly of polycations and polyanions is a promising approach for controlled loading and release in gene delivery. In this study, we have fabricated a series of multilayered films based on alternate deposition between positively-charged cationic phosphorylcholine copolymer (PC copolymer) and negatively-charged c-myc anti-sense oligodeoxynucleotide (AS-ODN). The growth of film thickness and increase of ODN loading capacity were monitored by spectroscopic ellipsometry (SE) and confocal laser scanning microscopy (CLSM). After elution into PBS buffer under physiological conditions, the elution profile was monitored by UV spectrometry and gel electrophoresis. Employing a secondary transgenic vector, the cellular uptake of the eluted AS-ODN into HeLa cells was evaluated by fluorescent microscopy and FACS analysis. The biological effect of eluted AS-ODN was evaluated by cell growth inhibition. The results showed that AS-ODN loading capacity increased almost linearly with the number of PC polymer/ODN bilayers and was also strongly dependent upon the cationic charge density. Through swelling, a non-degradable release mechanism, the AS-ODN release was characterized by two distinguishable release regimes: a fast release regime during the first 6 hour period and a slow release regime from 6 hour to the 8th day, both of which were characterized by zero-order kinetics. Gel electrophoresis showed excellent DNA integrity and strong transfection was observed when the eluted ODN was transfected into HeLa cells. Cell growth was significantly inhibited by eluted AS-ODN, indicating its full bioactivity. These results demonstrate that PC multilayered polymer films are capable of delivering a prescribed amount of anti-sense ODN with a controllable kinetic profile and that the multilayer process is more efficient and reliable than most other existing coating approaches largely based on single-layer fabrication.