The purpose of this work is to design a well-defined cationic dextran for intravenous gene delivery into tumor and examine the effect of the dextran on transfection efficacy in vivo. To this end, disulfide-linked dextran–linear polyethylenimine (Dex–SS–LPEI) conjugates were designed as non-viral vectors for intravenous gene delivery into tumor-bearing Balb/c nude mice. By coupling different molecular weights (2 kDa or 5 kDa) of LPEI disulfide pyridine to thiolated dextran (5 kDa or 10 kDa), Dex–SS–LPEI conjugates were prepared which have dextran as the main chain and disulfide-linked LPEI as the side chain. Dex–SS–LPEI conjugates can condense gene into nanosized polyplexes with moderate surface charge. Besides, the polyplexes of the conjugates have an improved colloidal stability under physiological conditions as compared to that of 22 kDa LPEI lacking dextran and can liberate gene by disulfide cleavage in an intracellular reducing environment. In vitro transfection experiments manifest that Dex–SS–LPEI conjugates mediate efficient gene transfection in different cells. The most efficient transfection in vitro was found for the conjugate with 5 kDa dextran and 5 kDa LPEI. Besides, Dex–SS–LPEI conjugates are practical for systemic gene delivery into tumor-bearing Balb/c nude mice by intravenous injection, affording comparable or higher transgene expression in HepG2 or SKOV-3 tumor than that yielded by 22 kDa LPEI as a positive control. Further, the polyplexes of the conjugate with 10 kDa dextran and 5 kDa LPEI can induce comparable transgene expression in the tumor but lower expression in the lung when compared to those of 22 kDa LPEI. The results of this study indicate that dextran plays a critical role in regulating in vivo gene delivery properties of Dex–SS–LPEI conjugates and transfection efficacy in tumor-xenografted nude mice. Dex–SS–LPEI conjugates have low cytotoxicity in vitro and cause no death of the mice, showing great potential as safe and highly efficient gene delivery vectors towards cancer gene therapy.