In situ characterization of Surface denaturation of a protein was realized by newly developed interface-selective multiplex electronic sum frequency generation spectroscopy. The observed electronic spectra of cytochrome c at the air/water interface exhibited a broad feature, which demonstrated coexistence of the nativelike and denatured protein at the interface. This situation of the mixed conformation at the air/water interface did not change in the acidic condition of pH = 2 where the protein was completely denatured in the bulk water. In sharp contrast, only native spectrum was observed at the silica/water interface.