A variety of degradable hyperbranched poly(ester amine)s containing primary, secondary and tertiary amino groups, were synthesized and evaluated as non-viral gene carriers. The polymers were obtained in high yields through a Michael-type conjugate addition of diacrylate monomers with trifunctional amine monomers. Analysis of degradation products using liquid chromatography–mass spectroscopy (LC–MS) demonstrated that all poly(ester amine)s had a hyperbranched structure with a degree of branching of approximately 0.30. These poly(ester amine)s were readily water-soluble and degradable under physiological conditions (pH 7.4, 37 °C), in which more than 10% ester bonds were hydrolyzed within 4 h. Moreover, these hyperbranched poly(ester amine)s showed high buffering capacities between pH 5.1 and 7.4. Three out of nine synthesized polymers, i.e. p(HDDA-AEP), p(HDDA-AMP), and p(BDDA-AMP), were shown to effectively condense plasmid DNA into small-sized (not, vert, similar94–135 nm) and positively charged complexes. Polymer/DNA complexes (‘polyplexes’) based on these three polymers, and larger complexes of p(BDDA-AEP) (not, vert, similar497 nm) were able to transfect COS-7 cells in vitro. Importantly, the transfection activity of polyplexes was preserved in the presence of serum proteins. The highest transfection level was observed for p(HDDA-AEP) polyplex which had a transfection efficiency higher than or comparable to that polyplexes of polyethylenimine (PEI) and poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA). Furthermore, these poly(ester amine)s revealed no or low cytotoxicity. These results demonstrated that hyperbranched poly(ester amine)s can be applied as safe and efficient gene delivery polymers.