A novel amphiphilic biodegradable cationic hyperbranched poly(ethylene glycol)-polyethylenimine-poly(gamma-benzyl L-glutamate) (PEG-PEI-PBLG) block copolymer was successfully synthesized by ring-opening polymerization (ROP) of N-carboxyanhydride of gamma-benzyl-L-glutamate (BLG-NCA) with PEG-PEI as a macroinitiator. PEG-PEI was firstly prepared by coupling of PEG and PEI using hexamethylene diisocyanate (HMDI). The structural properties of PEG-PEI-PBLG copolymers were confirmed by 1H NMR and GPC. The copolymers were found to be self-assembled in water with critical micelle concentration (CMC) in the range of 0.00368-0.0125 g/l and high hydrophobic micelle core. The micelle size and CMC obviously depended on the hydrophobic block content in the copolymer and the ionic state of the PEI block. The CMC decreased with the increase in the PBLG block content. The decrease of micelle size and the increase of CMC simultaneously occurred with the protonated degree of PEI block by addition of HCl solution. ESEM and Gel retardation assay showed that the cationic micelles had ability to encapsulate plasmid DNA. The copolymer has potential medical applications in drug and gene delivery.