In order to estimate surface functions of biosensors, surface analytical tools such as time-of-flight secondary ion mass spectrometry (TOF-SIMS) are required for evaluating, identifying and quantifying the biochemical structures of biosensor surfaces. Surface of an optic immunosensor that uses the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by reactions between proteins, was investigated with TOF-SIMS for estimating and modifying the protein immobilization processes. Secondary ion images of TOF-SIMS show that protein distribution on the sensor surface is not homogeneous. The results indicate that the fluorescence background may be high when proteins are localized on specific area on the biosensor, because a part of immobilized proteins, covered with other proteins, are not able to contribute the reaction with immunoglobulin G (IgG). Thus the estimation of protein immobilization on the biosensor surface with TOF-SIMS clarifies the performance of the biosensor and will contribute to the development of biosensors.