Nanosized micelles based on cationic, amphiphilic poly(ethylene glycol)-poly(ω-pentadecalactone-co-N-methyldiethyleneamine-co-sebacate) (PEG-PPMS) block copolymers have been successfully developed to serve as a new type of biodegradable non-viral vectors for DNA delivery. PEG-PPMS copolymers with various compositions were synthesized in one step via lipase-catalyzed copolymerization of ω-pentadecalactone (PDL), N-methyldiethanolamine (MDEA) and diethyl sebacate (DES) with poly(ethylene glycol) methyl ether (MeO-PEG-OH). The effects of PEG molecular weight, PEG and PDL contents on the biological properties (including the gene transfection efficiency) of the copolymer micelles were investigated. The LucDNA-loaded micelles formed from the copolymers with 30-50 wt% PEG showed high stability in serum-containing aqueous medium, which is in sharp contrast to rapid aggregation of LucDNA/PPMS polyplex particles. The conjugation of PEG to PPMS chains significantly reduces the cytotoxicity and hemolysis activity of the PEG-PPMS micelles. Compared to PEG-free PPMS, the micelles of PEG-PPMS copolymers with optimal compositions (e.g., 42%PEG5K-PPMS-10%PDL and 42%PEG5K-PPMS-20%PDL) exhibited enhanced capability to condense and protect DNA. Although the optimized micelles showed comparable or slightly lower gene transfection efficacy than the reference PPMS in vitro, the efficiency of LucDNA/42%PEG5K-PPMS-20%PDL micelles in transfecting tumor cells in mice was twice as high as that of LucDNA/PPMS polyplex particles due to their strong DNA condensation ability and excellent stability under physiological conditions. The PEG-PPMS micelle system with improved properties is a family of potentially promising non-viral vectors for in vivo delivery of therapeutic genes to treat tumors.