This work presents the synthesis of new mercapto calix[4]arenes derivatives (4 and 5). These derivatives were capped on Fe3O4 magnetic nanoparticles and subsequently encapsulated with Candida rugosa through sol–gel method to furnish enc-4 and enc-5, respectively, to enhance catalytic activity and enantioselectivity of lipase for hydrolysis reaction of racemic flurbiprofen methyl ester. Catalytic activity and enantioselectivity of enc-4 and enc-5 were assayed at different pH and temperature conditions and it was found that the resultant encapsulated enzyme exhibited higher thermal and operational stabilities compared to the free lipase in which enc-5 showed the excellent rate of enantioselectivity (E = 176) for S-flurbiprofen better than free lipase (E = 137) at pH 7 and 35 °C for 48 h. The time study shows that enantioselectivity reached the maximum value of E = 244 after 72 h. Catalytic activity of these materials was hardly affected by 20 and 23% after five usages of enc-4 and enc-5, respectively.