Polyethylenimine (PEI) is one of the most characterized non-viral vectors. It can condense DNA in a good manner and achieve high transfection efficiency. Minicircle DNA (mc-DNA) is a novel kind of supercoiled DNA which is devoid of bacterial backbone. mc-DNA is superior to conventional DNA for its higher transfection effciciency and longer time-span. In this study, we combined PEI and mc-DNA in gene delivery system. We investigated the physicochemical and biochemical effects of this non-viral system and further explore its potential in tumor gene therapy. mc-DNA was obtained by recombination of parental plasmid in the presence of L-arabinose, and complexed with PEI. The results of transmission electron microscopy and scanning electron microscopy showed that the particles were spherical and homogeneous. Through gel retardation assay and MTT assay, we found that there were no obvious differences in binding capability of PEI to mc-DNA and plasmid DNA, as well as in cytotoxicity. The results of dynamic light scattering showed that the size of PEI/mc-DNA was about 68 nm, a slight larger than that of PEI/plasmid DNA. Furthermore, the tumor cells transfected with mc-GFP showed higher GFP expression level than that of conventional plasmid. The same results were achieved in the cells treated with tumor-suppressor gene pten, assayed by RT-PCR and Western blot. It indicates that the system of PEI/minicircle DNA is promising in gene transfer.