Redox-responsive cationic polymers have gained considerable attention in gene delivery due to low cytotoxicity and spatio-temporal release of DNA into the cells. Here, we reported the synthesis of reducible disulfide conjugated polyethyleneimine (1.8 kDa) (denoted as SS-PEI) and its application to transfer pEGFP-ZNF580 plasmid (pZNF580) into EA.hy926 cell. This reducible SS-PEI polymer was prepared by one-step polycondensation reaction of low molecular weight PEI with bis-(p-nitrophenyl)-3,3'-dithiodipropionate. The SS-PEI successfully condensed pZNF580 into nano-sized complexes (170 ± 1.5 nm to 255 ± 1.6 nm) with zeta potentials of 3 ± 0.4 mV to 17 ± 0.9 mV. The complexes could be triggered to release pZNF580 when exposed to the reducing environment of 5 mM dithiothreitol. Besides, the SS-PEI exhibited low cytotoxicity. In vitro transfection results showed that SS-PEI exhibited good transfection efficiency comparable to PEI25kDa. Thus, the SS-PEI could act as an reducible gene carrier with good transfection efficiency and low cytotoxicity.