To date, the synthesis of efficient and safe gene carriers with low toxicity and appreciable gene transfection efficiency has been the major hurdle associated with non-viral gene carriers. Herein, we synthesized three amino acid-based diblock copolymers comprising glycine-leucine, leucine-phenyl alanine and glycine-phenyl alanine group containing blocks. The synthesis of all the diblock copolymers was confirmed by FTIR, 1H NMR, DLS and GPC techniques. All the polymers showed a high positive zeta potential value that varies from 45 ± 1 mV to 56 ± 1 mV, and the hydrodynamic size of the polymers varies from 250 ± 8 to 303 ± 14 nm. The three polymers showed negligible cytotoxicity compared with PEI (25 kDa) for MDA-MB-231 and NKE cells. Among all other polymers, P(HGN)n-b-P(HPN)m exhibited the highest biocompatibility with ∼70% cell viability at a concentration of 200 μg mL-1. Hemolysis data revealed that among all three polymers, P(HGN)n-b-P(HPN)m exhibited the highest blood compatibility, while up to a high concentration of 200 μg mL-1, it showed a very negligible amount (∼18%) of hemolysis. Most importantly, excellent gene complexation capability and good protection of pDNA against enzymatic degradation were observed with all three diblock copolymers. Interestingly, P(HGN)n-b-P(HPN)m/pDNA complex showed the smallest particle size (∼15 nm) and highest positive zeta potential as observed from TEM micrographs and DLS analysis, which probably results significantly higher level of cellular uptake and hence the highest transfection efficiency (∼85%) against MDA-MB-231 cells. Therefore, the diblock copolymer P(HGN)n-b-P(HPN)m with superior gene transfection efficiency in triple negative breast cancer may be an efficient non-viral vector for successful TNBC therapy in the future.