Surface modification by chemical reagents is of profound importance to modulate the surface characteristic and functionality of materials, which has attracted tremendous interest in many research fields and industrial applications, such as froth flotation of minerals. In this work, a new reagent S-[(2-hydroxyamino)-2-oxoethy1]-O-octyl-dithiocarbonate ester (HAOODE) with heterodifunctional ligands was designed and synthesized to improve the flotability of chalcopyrite (CuFeS2). Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy results showed the co-adsorption of heterodifunctional ligands (i.e., dithiocarbonate and hydroxamate groups) of HAO ODE on chalcopyrite via Cu(I)-S and Cu(II)-O covalent bonds. The bubble probe atomic force microscopy (AFM) technique was employed to quantitatively measure the air bubble-chalcopyrite interactions with and without the reagent adsorption. AFM force results revealed that the bubble could be more readily attached to flat chalcopyrite after HAOODE treatment under different hydrodynamic conditions because of the enhanced hydrophobic interaction, with the decay length D-0 increasing from 0.65 to 1.20 nm. The calculated bubble-particle interaction forces also demonstrated the critical influence of HAOODE treatment, hydrodynamic conditions, and bubble size on the interaction behavior and thin-film drainage process in flotation. In froth flotation, HAOODE exhibited superior recovery for chalcopyrite over pH 3-12 and excellent selectivity for chalcopyrite against pyrite (FeS2) above pH 10.5, as compared to the conventional reagent sodium isobutyl xanthate. This work provides a useful approach to develop effective reagents that could selectively adsorb on desired mineral surfaces through heterodifunctional ligands and to quantitatively evaluate the role of reagent adsorption in the interactions between air bubbles and mineral surfaces at the nano-and microscale. Our results show implications on developing molecular design principles of novel reagents for surface modifications of materials in a wide range of engineering and biological applications.