Flotation separation of galena and chalcopyrite is always a difficult problem in mineral processing. In this paper, the selective preoxidation of galena and chalcopyrite with sulfuric acid was developed, and then the two minerals were completely separated by flotation. The surface oxidation mechanism of galena and chalcopyrite with sulfuric acid was analyzed by Fourier transform infrared spectroscopy (FT-IR) and Atomic Force Microscopy (AFM), and the results showed that hydrophilic oxide film was formed on the galena surface, while the surface of chalcopyrite is still hydrophobic sulfide film, which led to the separation of the two minerals by flotation. In addition, the Response Surface Methodology (RSM) was used to analyze the influence of main preoxidation parameters on the flotation separation of copper-lead concentrate, and the parameters were further optimized, as follows: sulfuric acid concentration of 5.3 mol/L, oxidation temperature of 101.8 °C and time of 48.3 min. The mixed concentrate containing Cu 11.57% and Pb 16.75% was preoxidized under the above conditions, and the flotation separation verification results showed that Cu concentrate with Cu grade of 18.09% and recovery of 95.41%, and Pb concentrate with Pb grade of 44.96% and recovery of 95.94% was obtained respectively. This paper provides a new method of preoxidation combined flotation to achieve high-efficiency separation of copper-lead mixed concentrate.