We synthesized the amine-functionalized Zr-MOF (UiO-66-NH2) to provide a covalent binding site for L-proline in the presence of EDC and NHS. Obtained proline-substituted Zr-MOF (UiO-66-NH/Pro) was used as a new carrier for the immobilization of Candida rugosa lipase. To compare catalytic activity, enantioselectivity and stability of the lipase-immobilized UiO-66-NH/Pro (UiO-66-NH/Pro/CRL), lipase was also immobilized on the surfaces of UiO-66-NH2 in the presence of L-proline by physical adsorption to afford UiO-66-NH2/Pro/CRL(enc.). The FTIR, TGA, SEM, EDX, ζ potential and PXRD were performed to assess the structures of the lipase-immobilized Zr-MOFs. Moreover, UiO-66-NH/Pro/CRL and UiO-66-NH2/Pro/CRL(enc.) were successfully used in the hydrolysis reaction of p-nitro-phenylpalmitate (p-NPP). The influence of pH and temperature was also examined. In the optimized conditions, UiO-66-NH/Pro/CRL exhibited higher catalytic activity and enantioselectivity than UiO-66-NH2/Pro/CRL(enc.) in the hydrolysis (UiO-66-NH/Pro/CRL: 64 U/g of activity and 254 of enantioselectivity (E); UiO-66-NH2/Pro/CRL(enc.): 53 U/g of activity and 244 of enantioselectivity (E) reaction. Lineweaver-Burk kinetic studies demonstrated that Vmax value for UiO-66-NH/Pro/CRL was obtained to be higher, whereas, it showed lower Km than UiO-66-NH2/Pro/CRL(enc.). Furthermore, the residual catalytic affinities of UiO-66-NH/Pro/CRL and UiO-66-NH2/Pro/CRL(enc.) in the model hydrolysis reaction were found to be 80% and 60% after five cycles of reuse.