DNA supramolecular recognition is employed for the immobilization of 2D photonic crystals of monodisperse colloidal microspheres. Amine-terminated DNA oligomers are covalently attached to carboxy-decorated microspheres and substrates while preserving their colloidal stability and organization properties. Following a capillary-force-assisted organization of DNA-decorated microspheres into close-packed 2D opaline arrays, the first monolayer is immobilized by DNA hybridization. Various parameters affecting the long-range order of such opaline arrays are investigated, including surface hydrophobicity and the relative strengths of the specific versus nonspecific interactions. The type and concentration of salt and the process temperature are also optimized for the hybridization between microspheres and substrate. The selective removal of non-specifically bound multilayers is accomplished by carefully passing an air/liquid interface over these arrays. DNA hybridization was found to play an important role in immobilizing the first monolayer of 2D opaline arrays while preserving its long-range order, with an approximate binding strength three times higher than that of non-specific interactions.