Polyethyleneimne (PEI) is a very efficient transfecting agent but is toxic due to high charge density. To generate a vector which is efficient and less cytotoxic, PEI was conjugated with pullulan (PPEI). Further conjugation was done on PPEI with zwitter ionic betaine which possess antifouling property. PEI of molecular weight 1.2, 2, and 10 kDa were used in the study. Buffering capacity of pullulan-PEI-betaine (PPB) conjugates was found to be sufficient enough for the polymers to make endosomal escape. The polymers proved to be less cytotoxic and highly hemocompatible than PEI. Nuclear localization of YOYO tagged DNA was observed with the nanoplexes developed using PPEI and PPBs of PEI 10 kDa. Transfection efficiency was evaluated using p53 expressing gene and the live dead assay demonstrated very high transfection efficiency with PPB conjugates of PEI 10 kDa.