This paper presents a novel strategy for the simple immobilization of biomolecules on epoxy-functionalized supports via a one-pot three component reaction. Lipase B from Candida antarctica (CALB) and Rhizomucor miehei lipase (RML) as model enzymes were immobilized on epoxy-functionalized silica and mesoporous silica nanoparticles (SBA-15). Investigation of the mechanism of this reaction confirmed the participation of three functional groups including carboxylic acid groups from the enzyme surface, epoxy groups from the support and isocyanide from the reaction medium. The results revealed very rapid immobilization of 10 and 40 mg of RML on 1 gr of the supports shortly after 30 minutes of incubation. The loading capacity of the supports was also dramatically improved with the maximum loading of 158 mg of CALB and 77 mg of RML on SBA-15 and silica, respectively. Silica-epoxy-RML showed an enantiomeric excess (ee) of 92% and an E-value of 29.9 in the enantioselective esterification of (R,S)-ibuprofen.