Now days, study on protein analysis has accomplished to understand for biomolecular interaction. But, when protein is analyzed, protein is limited to amplify. Thus micro detection system of protein is demanded in protein analysis in biomolecular. This paper introduce to micro detection of protein using spectral analysis of reflection interference. An optical biosensor using reflection interference is a non-destructive, label-free, and versatile technique in liquid and gaseous phase, that is well suited for monitoring biomolecular interactions. To increase sensitivity, this paper is different from previous method for reflection interference measurement using front side incidence. And this paper uses an antigen-antibody interaction to measure sensitively the specific protein. Biological material deposited at the surface during binding event increases the optical thickness of the interference layer. Increment of the optical thickness related to interference pattern change. In this paper, the optical thickness is calculated through spectral analysis using model fitting. The results show that thickness for antibody is about 12nm and thickness for antibody and antigen is about 19nm. Finally, the micro detection of protein is detected by the calculated optical thickness.