It will form protein corona on the surface of nanoparticles when exposed to the biological environment. The process of protein corona formation is crucial in biomedical engineering, nanomaterials development and research, especially the protein corona formation process of medical engineering materials in blood samples, and there is no mature method for real- time monitoring. Silica colloidal crystal films were prepared by vertical deposition on the glass slides. The glass slides were placed vertically in an ethanol suspension of silica nonospheres with diameters of 110, 190 and 280 nm. After 7 days of ethanol evaporation, a large area of silica colloidal crystal film was formed on the surface of the glass slide. The glass slides of silica colloidal crystal films were assembled into reaction cell, and the reflectometric interference spectroscopy (RIfS) system assembled by microscope light source and spectrometer were used for detection and optical thickness as a parameter. And different concentrations of hydrochloric acid were used to test the performance of the RIfS system. In this study, RIfS was utilized to detect the protein corona formation of human serum albumin, human fibrinogen and immunoglobulin G, as well as blood samples by silica colloidal crystal films which have special optical properties. It was found that the formation of immunoglobulin G had the highest optical thickness variation due to its conformation. According to the formation process of human serum albumin on silica nanosheres with different diameters, the optical thickness of protein corona was also higher on the of silica nanospheres with higher curvature. In addition, through the detection of plasma and whole blood samples, it was found that the silica colloidal crystal films could basically eliminate the interference of blood cells to the detection of protein corona. This work developed a real-time unlabeled protein corona research method, which provides a basis for the clinical application of biomedical materials.