Polyurethanes (PUs) are a class of biodegradable polymers that have been applied as tissue-engineering materials with minimum toxicity. In our study, a new series of cationic PUs containing tertiary amines in the backbone and primary, secondary and tertiary amines in the side chains (PU1, PU2 and PU3, respectively) was synthesized and used as nonviral vectors for gene delivery. In addition, we introduced glycidol into the structure of PU for greater solubility and biocompatibility and grafted various amines in the side chains (PUg1, PUg2, PUg3). The structural characteristics of PUs and the physicochemical properties of their formed complexes with DNA were determined and correlated with their transfection efficiency. The results reveal that PU1, PU3, PUg1 and PUg3 could bind with DNA and yielded positively charged complexes with a condensed size required for transfection. These PUs are considered to be noncytotoxic (hundred times less) compared to polyethylenimine (PEI) or poly(2-dimethylaminoethyl methacrylate), (PDMAEMA). The hydrolytical degradation studies indicate that PU-glycidyl systems (PUg1 and PUg3) can be degraded in 20 mM HEPES buffer at pH 7.4 in approximately 8 h but that PU1 and PU3 lasted much longer. PUg1 and PUg3 are the best amongst cationic PUs to transfect DNA into COS-7 cells with an efficacy comparable to the well-known gene carrier PDMAEMA. In addition, PUg1 and PUg3 possess greater biocompatibility and biodegradability. A new way to prepare cationic polymers without cytotoxicity but with highly transfecting activity could be very helpful to the in vivo gene transfection where large amounts of cationic polymers are required.