The influences of environmental factors on how PEI derivatives mediate gene delivery are absolutely essential to obtaining high transfection efficacy, but have been rarely investigated until now. In this study, biotinylated PEI-SS/avidin bioconjugate (ABP-SS) mediated gene transfection in HepG2 cells was exposed to variying environmental factors, such as pH, NaCl, serum, temperature and time. Physicochemical characteristics of ABP-SS/DNA complexes were evaluated in terms of agarose gel electrophoresis, particle size measurements, and in vitro transfection assays. The DNA binding ability of ABP-SS was weakened when pH value was decreasing. It was inferred that ABP-SS/DNA complexes could form compact structures at pH 7.4 and looser structures at lower pH values. Transfection efficiencies were largely dependent on the pH of the culture medium, and the optimal pH of culture medium for ABP-SS mediated gene delivery in HepG2 was 7.4. The particle sizes of ABP-SS/pDNA complexes formed in 150 mM NaCl solution were less than 280 nm, and were larger than those of complexes formed in deionized water. The transfection ability of ABP-SS/pGL-3 complexes formed in deionized water was much weaker than that formed in 150 mM NaCl solution. The presence of lower serum contents had no obvious effect on the transfection efficiency of ABP-SS/pGL-3 complexes. The optimum complex-forming temperature and time for ABP-SS/pGL-3 complexes were 37 °C and 30 min. Cell morphology was observed by live cell confocal microscopy. It can be concluded that the cytotoxicity of ABP-SS in HepG2 cells was the result of apoptosis, and naked DNA had no negative impact on cells.