Owing to their tailorable structure deficiencies and intrinsic framework robustness, Zr−MOF membranes hold great promise in energy-efficient separation of diverse industrially important gas mixture, while their complete room-temperature (RT) production is extremely advantageous for large-scale application. Herein, we reported a facile complete RT protocol for preparing (111)-oriented UiO-66 membranes with CPO 111/002 of 7.4 on ZrO 2 buffer layer-modified porous α-Al 2 O 3 substrate with a Zr(n-OPr) 4 source. Experimental results indicated that carrying out the reaction at RT resulted in higher missing-linker numbers in the UiO-66 framework (1.5 per Zr 6 formula unit) and therefore higher CO 2 /N 2 adsorption selectivity, attributing to higher affinity interaction between CO 2 and defective sites in Zr 6 -oxo cluster nodes. Benefiting from high linker deficiencies and low intercrystalline defects, the ideal selectivity of CO 2 /N 2 (36.5) on the prepared membrane was superior to the majority of polycrystalline MOF membranes measured under comparable conditions; moreover, unlike other hydrophilic membranes, our membrane manifested steady CO 2 /N 2 separation performance under moisture conditions. The above reproducible protocol provides an appealing solution for alleviating residual concerns hindering the large-scale production of high-performance Zr−MOF membranes.