A novel surfactant, thiocarbonohydrazide (TCH), was synthesized and tested for the first time as a selective chalcopyrite depressant in Cu-Mo separation. Its adsorption mechanisms on chalcopyrite were studied by flotation tests, zeta potential, FTIR, XPS and ToF-SIMS measurements. FTIR and zeta potential analyses suggested that there was a strong chemisorption reaction between TCH and chalcopyrite, resulting in the formation of TCH–copper complexes. XPS and ToF-SIMS measurements further confirmed the chemisorption of TCH onto the chalcopyrite surface and showed that this chemisorption reaction is due to its S and N atoms, which form five-membered chelating rings by releasing H ions.