Multispectral imaging is becoming a new powerful tool in a wide range of biological studies by adding spectral, spatial, and temporal dimensions to tissue abnormity and the underlying biological processes. Conventional spectral imaging systems are bulky, expensive, require multiple exposures, or extensive post-processing to align multiple images of pure spectral components. Recently, a break-through technology has emerged to instrument multispectral imaging technology into handheld real-time devices using miniaturized filter mosaic containing densely patterned micro-arrayed multiple channel bandpass optical filters. The filter mosaic can be directly placed near the focal plane, immediately in front of the imaging sensor of an off-shelf charged-coupled device/complementary metal–oxide–semiconductor camera, with the potential of one element of such a micro-arrayed filter to cover one pixel of the imaging sensor. This paper reveals the technical details of how such a micro-arrayed multichannel optical filter is fabricated using traditional multifilm vacuum deposition and the modern microlithography technologies. The selection of different coating materials, their structures, and effects to the adhesive forces between film and substrate, the spatial resolution, width of passing band, and the transmittance of the resulting miniaturized optical filter are discussed.