Polyethylenimine (PEI) is one of the most promising polymeric gene vectors, however its applications are limited by serious cytotoxicity and moderate transfection efficacy. Fluorination is an efficient strategy to improve the transfection efficacy of cationic polymers while reducing their cytotoxicity. Here we grafted different fluoroalkyl chains to PEI via oxirane and anhydride reactions. The fluorinated PEIs show superior transfection efficacy on both 2D and 3D cell cultures to unmodified PEI. These fluorinated polymers allow efficient gene transfection at relatively low nitrogen to phosphorus ratios and thereby ensure low cytotoxicity on the transfected cells. Fluorinated PEIs prepared via the oxirane reaction are much more stable in aqueous solutions than the ones prepared by the anhydride reaction and show reproducible gene transfection during a period of 6 months. This study extends the applicable scope of fluorination on improving the transfection efficacy of polymers and generates a list of gene vectors for efficient 2D and 3D cell culture gene transfection.