Linear fluorinated polyamides with reversible cationic charges are feasibly prepared to be used as highly efficient gene vectors in HEK293 cell line. Due to the uniform polymer structure, the relationship between the physicochemical properties and transfection efficiency could be unambiguously investigated. The different efficiency in the application of gene delivery between the parent polyethylenimine (PEI) and the polyamides is directly associated with the differences in chemical and physical properties between secondary amines and fluorinated amides. We found that fluorination not only increases the cellular uptake of polymer/DNA polyplexes, but it also decreases cytotoxicity in terms of inducing lower concentrations of proinflammatory cytokine TNF‐α. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2132–2139, 2019.