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In this work, a novel multispectral sensing system consisting of nanostructured filter matrix and a charge-coupled device (CCD)-based image sensor has been developed to overcome the limitation of the conventional pigment filtered sensors, which are difficult to be fabricated at a microscale and usually showing a pronounced degradation. By designing the filters in guided-mode resonance (GMR) architecture, light transmission efficiencies of ~90% with low sidebands and sharp peaks can be obtained, which are critical characteristics for realizing precise optical measurement systems. To optimize the transmission functions, various materials and structural parameters have been simulated. Electron beam nanolithography is employed in the device fabrication to fabricate pixel-wise independent filter functions. After being characterized in terms of their wavelength filtering capability, the developed GMR filters are then combined with image sensors, particularly for addressing biological applications.