To overcome the different extra-/intracellular barriers in gene delivery, tumor-targeted and pH/redox-responsive ternary polyplexes with charge-conversional properties were prepared through a modular self-assembly strategy. Firstly, the thiolated trimethylated chitosan (TMC-SH) was synthesized to crosslink and condense pDNA through electrostatic interaction and disulfide formation, which obtained the TMC-SS/pDNA binary polyplexes with redox-responsive gene release. To further endow the binary polyplexes with tumor targeting and endo/lysosomal pH-triggered charge-reversal properties, a folate conjugated cis-aconitic amide-polyethylenimine (FA-PEI-AcO) was synthesized to shield the positive TMC-SS/pDNA, generating the FA-PEI-AcO/TMC-SS/pDNA ternary polyplexes with a size of ~190 nm and negative surface-charges. The ζ-potential of the polyplexes was stable at physiological pH and increased rapidly from -14 mV to + 20 mV at pH 5.5 (endo/lysosomal pH) due to the breakages of acid-liable amide bonds and the subsequent de-shielding of FA-PEI-AcO layers, which might benefit the endo/lysosomal escape of the polyplexes. Afterward, the polyplexes could redox-responsively release gene at higher intracellular concentrations of glutathione. By taking advantage of such multi-responses, significantly enhanced transfection efficiency was achieved in vitro in Hela cells for the ternary polyplexes. These results suggested that the newly developed polyplexes had potential application for gene delivery.