Fluorinated biomaterials have been reported to have promising features as non-viral gene carriers. In this study, a series of fluorinated polymeric gene carriers were synthesized via Michael addition from low molecular weight polyethyleneimine (PEI) and fluorobenzoic acids (FBAs)-based linking compounds with different numbers of fluorine atoms. The structure-activity relationship (SAR) of these materials was systematically investigated. SAR studies showed that fluorine could screen the positive charge of these polymers. However, this shielding effect of fluorine would endow fluorinated polymers with good balance between DNA condensation and release. In vitro transfection results suggested that these fluorinated polymers could mediate efficient gene delivery. Flow cytometry and confocal microscopy studies demonstrated that more efficient cell uptake could be achieved by fluorinated materials with more fluorine atoms. Cytotoxicity assays showed that these fluorinated materials exhibited very low cytotoxicity even at high mass ratios. This study demonstrates that FBA-based fluorinated biopolymers have the potential for practical application.