In this paper, 4-amino-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one (ATDT) was synthesized and introduced as a depressant for selective flotation separation of molybdenite from chalcopyrite. Its flotation performance and adsorption mechanism on minerals were first investigated by flotation, UV spectra, zeta potential, Fourier Transform Infrared Spectroscopy (FTIR), and X-ray Photoelectron Spectroscopy Measurements (XPS). The bench scale tests indicated that ATDT exhibited stronger depressing power than chalcopyrite, and the selective index of Mo/Cu improved significantly in the presence of ATDT. The results of the UV spectra, zeta potential and FTIR demonstrated that ATDT chemisorbed on the chalcopyrite surface. The XPS results further confirmed that ATDT might chemisorb onto the chalcopyrite surface through S and N atoms to form five-membered chelate rings and a postulated adsorption mode was presented. For molybdenite, the different measurements agreed well with each other and implied that ATDT might weakly physisorb onto a molybdenite surface.