Alginate has been previously utilized as an adjuvant in nanoparticle-mediated gene delivery. In this study, we investigated the extent to which alginate promotes in situ transfection from polyelectrolyte multilayers (PEMs). After spiking free alginate molecules, DNA release from PEMs was increased and transgene expression was enhanced, suggesting that alginate may compete with DNA to weaken electrostatic interaction within PEMs. Consequently, alginate was applied to compose multilayers with DNA and polyethyleneimine (PEI). Interestingly, the incorporated alginate increased not only deposition but also delivery of DNA from PEMs. Quartz crystal microbalance (QCM) analysis suggested that the deposited alginate enhanced PEI adsorption which in turn augmented subsequent DNA deposition. In situ transfection experiments revealed that alginate within PEMs improved not only the level but also the duration of transgene expression. This system should be a potential strategy to regulate gene delivery from scaffolds for tissue engineering application.