Efficient DNA delivery is essential for introducing new genes into living cells. However, effective virus-based systems carry risks and efficient synthetic systems that are non-toxic remain to be discovered. The bottle-neck in synthetic systems is cytotoxicity, caused by the high concentration of DNA-condensing compounds required for efficient uptake of DNA. Here we report a polyethyleneimine (PEI) grafted ultra-small graphene oxide (PEI-g-USGO) for transfection. By removing the free PEI and ensuring a high PEI density on small sized graphene, we obtained very high transfection efficiencies combined with very low cytotoxicity. Plasmid DNA could be transfected into mammalian cell lines with up to 95% efficiency and 90% viability. Transfection in zebrafish embryos was 90%, with high viability, compared to efficiencies of 30% or lower for established transfection technologies. This result suggests a novel approach to the design of synthetic gene delivery vehicles for research and therapy.