A mild chemical procedure for the derivatization of PMMA as the isocyanate on rigid supports is described. The proposal is based on spin-coating the support with a hydroxy-modified PMMA polymer, followed by treatment with (3-isocyanatopropyl)triethoxysilane, developing an isocyanate-ended PMMA. Oligonucleotide hybridization assays performed on this surface demonstrate that the process is simple and highly effective and agree with the results of other modified materials, including aminated PMMA, gold, and glass. As a demonstration, it has been applied to covalently attached aminated oligonucleotides on a modified audio-video compact disc (CD) surface to perform DNA probe hybridization assays. Measurements were carried out with a CD player, by detecting the solid microspots precipitated from an enzymatic reaction (HRP-TMB). This new approach, being a competitive technique, opens a broad horizon of applications for point of care or in situ needs.