A series of thioamide surfactants including thio-n-butyramide (TnBA), thiohexanamide (THA), thio-p-toluamide (TpTA) and thio-N-acetylaniline (TNAA) were synthesized and used as flotation collectors for selective separation of chalcopyrite from pyrite. The micro-flotation results demonstrated that compared with TnBA and TNAA, TpTA and THA possessed better collecting ability to chalcopyrite, meanwhile, TpTA shown better selectivity than THA. After treatment by the four collectors, the contact angles of chalcopyrite improved in different degrees, among which TpTA improved the greatest. UV/vis spectra illustrated that TpTA can selectively react with Cu+ or Cu2+, not with Fe2+ or Fe3+. Adsorption thermodynamics and kinetics elucidated that TpTA adsorbed onto chalcopyrite surfaces was a spontaneously endothermic chemisorption process. FTIR, XPS spectra and DFT calculations gave evidences that Cu atoms might bond with S and N atoms of TpTA by formation of Cu-S and Cu-N bonds. Density functional theory (DFT) and octanol-water partition coefficient (C log P) further revealed that TpTA possesses excellent hydrophobicity and donating electron ability to chalcopyrite.