A group of new disulfide-containing poly(β-amino ester)s was synthesized and evaluated as non-viral gene delivery vectors. These linear polymers were obtained by Michael addition of a number of selected amines to bis(2-acryloyloxyethyl) disulfide. It was shown that the disulfide-containing poly(β-amino ester)s can condense plasmid DNA into nanoscaled and positively charged polyplexes, which could be rapidly destabilized in an intracellular reductive environment. Polyplexes of the polymer of histamine and bis(2-acryloyloxyethyl) disulfide show efficient transfection of COS-7 cells along with a relatively low cytotoxicity.