The invention discloses a method for enhancing the separation of cysteine molecules through doping of gold surface. The method comprises: obtaining a pure Au(532) surface from a gold bulk phase, replacing the uppermost layer atom on the Au(532) surface with Ag, setting the obtained surface as an Ag/Au(532) surface, constructing L-chiral cysteine molecules and D-chiral cysteine molecules, and optimizing the structures; respectively adsorbing the optimized L-chiral cysteine molecules and the optimized D-chiral cysteine molecules to different positions of the optimized Au(532) surface and the optimized Ag/Au(532) surface, optimizing the structures, and respectively finding the most stable configurations; and carrying out difference on the adsorption energies of the L-chiral cysteine moleculesand the D-chiral cysteine molecules on the Au(532) surface and the adsorption energies of the L-chiral cysteine molecules and the D-chiral cysteine molecules on the Ag/Au(532) surface to respectivelyobtain [delta]E1 and [delta]E2, wherein the more the [delta]E, the more significant the desorption sequence of the L-chiral cysteine molecules and the D-chiral cysteine molecules, and the stronger the separation. According to the present invention, the accurate DFT calculation method and the rational design are used, and the cysteine isomer distinguishing ability of the bimetallic surface obtained by doping Ag on the Au(532) surface is increased by 45% compared with the pure gold surface.