Cationic polymer based non-viral vector has been considered a promising approach for gene delivery. How to achieve a vector with high transfection efficiency along with low cytotoxicity is still under development. Consequently, significant research has been conducted to predict the internalization mechanism of biopolymer-DNA polyplex which influences the transfection efficiency. The transfection pathway that a cationic vector starts with the cellular entry and the capacity of polyplex that avoids lysosomes degradation are two key factors influencing the transfection efficiency. The degradation of the polyplex in lysosomes thus limits the transfection efficiency. To overcome this problem, we developed a bovine serum albumin-oligochitosan(BSA-OC) biopolymer as a non-viral vector for DNA delivery. We chose CHO-K1 and HEK293T cells in order to determine the internalization pathways of the BSA-OC/pDNA polyplex. The cells were treated with different endocytic inhibitors to study the specific pathways. The fact that sodium azide inhibited up to 40% transfection efficiency suggested the endocytosis pathway played a major role in cellular uptake of the polyplex. When a caveolae-mediated pathway was blocked by genistein, GFP expression was significantly reduced. Meanwhile, treatment with methyl-β-cyclodextrin decreased the transfection efficiency about 30% to 60% indicating the involvement of membrane cholesterol in the endocytotic process. Furthermore, chlorpromazine and monensin enhanced the transfection efficiency, suggesting that clathrin-mediated pathway and lysosome pathways did not play a significant role. Finally, a combination of Lysotracker and FITC-labeled biopolymer revealed that the polyplex was not co-localized with Lysotracker and was able to escape from lysosomes. Our results suggested that the caveolae-dependent internalization of hybrid biopolymer and the avoidance of lysosomes degradation enhanced the transfection efficiency to 90% in both cell lines.