In recent years, label-free biosensors not requiring external modifications have been receiving intense attention. A label-free optical biosensor, which retains many of the desirable features of conventional surface plasmon resonance (SPR) reflectometry, namely, the ability to monitor the kinetics of biomolecular interactions in real-time without a label has been developed with several important advantages: the biosensor device is easy to fabricate, and simple to implement, requiring only an UV–Vis spectrophotometer or flatbed scanner. Importantly, the label-free optical biosensor can be easily multiplexed to enable high-throughput monitoring of biomolecular interactions in an arraybased format. In this research,the development of a localized surface plasmon resonance (LSPR)-based label-free optical biosensor using a surface modified nanoparticle layer is aimed. This optical detection method promises to offer a massively parallel detection capability in a highly miniaturized package. The two-dimensional nanoparticle layer was formed by the surface modified silica nanoparticles. The optical properties and surface analysis of nanoparticle layer substrate were characterized through transmissionmeasurements and atomic force microscopy (AFM). Simultaneously, the nanoparticle layer substrate was applied to the optical LSPR-based biosensor for label-free monitoring of the antigen–antibody reaction. The anti-fibrinogen antibody wasimmobilized onto the nanoparticle layer substrate surface. Different concentrations of fibrinogen were introduced to the anti-fibrinogen antibody immobilized nanoparticle layer substrate surface, and the change in the absorption spectrum, caused by the antigen–antibody reaction, was observed. By using this anti-fibrinogen antibody immobilized nanoparticle layer substrate; the detection limit of this optical LSPR-based biosensor was 10 ng/ml.