In this study, layer-by-layer (LbL) assembly of polyelectrolyte was facilitated using electric field assistance (EFA). To elucidate the EFA effects on polyelectrolyte multilayers (PEMs), the electric fields were solely administrated to either chitosan or DNA adsorption. Both DNA and chitosan adsorptions can be augmented under low electric field due to the electrophoretic deposition. However, the ensuing electrochemical reactions on electrode interfered with interactions between multilayers when the bias was larger than 0.5V, leading to retard deposition. Subsequent delivery experiments indicated that EFA during DNA deposition demonstrated superior DNA release. In contrast, no obvious improvements were observed for the groups using the EFA during chitosan deposition. Moreover, water contact angle experiments revealed different effects of electric fields on multilayer structure. Using EFA during DNA deposition led to a layered-form composition, whereas interpenetration of electrolytes was enhanced with the application of the electric field during chitosan deposition. For in vitro experiments, EFA during DNA deposition significantly enhanced in situ transfection performances of PEMs that the transgene expression levels were increased and the periods were extended, suggesting this method is potential to quantitatively and temporally improve substrate-mediated gene delivery.