Flotation collectors with strong collecting ability and superior selectivity have important application prospects for recovering complex and refractory copper sulfide resources. Herein, a novel surfactant of N-benzoyl-N’,N’-diethyl thiourea (BDETU) with CO and CS groups is synthesized for separation of chalcopyrite and pyrite. The flotation performance and adsorption mechanism of BDETU to chalcopyrite and pyrite were evaluated by flotation experimental, adsorption experimental, contact angle measurements, UV–vis spectroscopy, as well as FTIR analyses and DFT calculation. By comparing the flotation performances with the traditional flotation collector of O-isopropyl-N-ethyl thionocarbamate (IPETC), it becomes evident that BDETU exhibit stronger collecting ability and excellent selectivity for chalcopyrite against pyrite. These results might be attributed to the facts that BDETU can selectively adsorb on the surface of chalcopyrite and strengthen its surface hydrophobicity, while the hydrophobicity of pyrite surface is not improved with the weak adsorption of BDETU on its surface. The analyses of adsorption mechanism reveal that BDETU is chemically adsorbed on the surface of chalcopyrite by the reactions of the functional groups (CO and CS) to the copper species with the formation of COCu and CSCu structures.