Targeting of non-viral gene vectors to liver cells could offer the opportunity to cure liver diseases. In this paper, disulfide-containing polyethylenimine (PEI-SS) was synthesized from low molecular weight branched PEI and cystamine bisacrylamide (CBA), and then grafted with biotin. The obtained biotinylated PEI-SS was bioconjugated with avidin via the biotin-avidin interaction to form a novel gene vector, biotinylated PEI-SS/avidin bioconjugate (ABP-SS). Characteristics of ABP-SS and its pDNA complexes were evaluated in terms of acid-base titration, agarose gel electrophoresis, SEM morphology observation, particle size and ζ-potential measurements, and PEI-SS was used as the control. The acid-base titration results showed that ABP-SS exhibited comparable buffer capability to 25 kDa PEI. The results of gel electrophoresis indicated that ABP-SS was able to condense pDNA efficiently at an N/P ratio of 6 and could be degraded by reducing agent DTT. The ABP-SS/pDNA complexes had a mean particle size of 226 ± 40 nm and surface charges of 25 mV. The SEM images showed that the complexes had compact structures with spherical or quadrate shapes. In vitro cell viability and transfection of ABP-SS and PEI-SS were compared in HepG2, 293T and H446 cells. Among the three different cell lines, compared with PEI-SS, ABP-SS exhibited much lower cytotoxicity and higher transfection efficacy in HepG2 cells due to the biocompatibility of avidin and the specific interactions between avidin and HepG2 cells. Molecular probes were used to reveal the cellular uptake of complexes, and the results demonstrated that ABP-SS contributes to more cellular uptake of complexes in HepG2 cells, which was consistent with the transfection results.