In this study, a new application method for SERS named "reversed reporting agent" method is proposed for selective detection of biomolecules with a thiol group. In this method reporting agents such as rhodamine 6G (R6G) are capped on surfaces of silver colloidal nanoparticles. Analytes having a thiol group will replace the positions of reporting agents due to the strong interactions between silver and the thiol group, and then the SERS intensity of the reporting agents will be reduced. By monitoring the difference in the SERS intensity of reporting agents before and after the replacement of analytes, the concentration of the analytes can be estimated. To demonstrate the feasibility of this proposed method, glutathione was used as the analyte and R6G, crystal violet (CV), and thiacyanine (TC) were employed as the reporting agents, and factors that affect the capability of the determination of glutathione were investigated. The results indicate that R6G is the most suitable reporting agent, and the concentration of the reporting agents affects the sensitivity and selectivity of glutathione determination. Under the optimal conditions, the detection of glutathione can be finished within 20 minutes and the detection limit of ca. 1 microM can be achieved. Furthermore, the proposed method holds specific selectivity towards glutathione, which means it has possible practical applications.