Polyethyleneimine is one of the most efficient non-viral gene delivery agents. However, some limitations such as non-specific cell binding, interactions with blood components, and relatively high cytotoxicity restrict its in vivo applications. In order to improve the properties of this molecule as a gene transfection vector, in this study, we developed a nano-carrier system based on polyethyleneimine derivatives synthesized by grafting bi-functional polyethylene glycol molecules (MAL-PEG 3500-NHS) to 25 kDa branched polyethyleneimine polymer at three different molar ratios of 10, 20, and 30 (polyethylene glycol:polyethyleneimine). Variable domain of heavy-chain antibody against human epidermal growth factor receptor 2 as targeting agent was conjugated to polyethylene glycol-polyethyleneimine copolymer. In order to find the most effective resultant conjugate, the effect of these modifications on physicochemical properties, cytotoxicity, cellular uptake, and gene transfection efficiency of polyethyleneimine polymer was evaluated. According to the findings of this study and compared to unconjugated polyethyleneimine, PEGylated polyethyleneimine copolymers exhibit lower in vitro cytotoxicity. Polyethylene glycol-polyethyleneimine copolymer conjugated at a molar ratio of 10:1 (polyethylene glycol:polyethyleneimine) demonstrated a low cytotoxicity effect and desirable physicochemical properties. Anti-human epidermal growth factor