A kind of step optical biological probe was designed. The rigorous coupled wave analysis (RCWA) method was used for calculating reflection efficiency of the biological probe in reflectance spectroscopy. Results of numerical simulation indicated that between index of refraction of detecting samples and angle of incidence was linear in a certain peak value of the biological probe in reflectance spectroscopy. Moreover, relation between reflection efficiency of the biological probe and index of refraction of detecting samples was left Gauss curve. The new technology and results based on the study could be applied for some field of biosensors and biological analysis.