Two-dimensional (2-D) metal-organic frameworks (MOFs) combine the tunable structure and chemical functionality of MOF materials with the high aspect ratio of the 2-D morphology, which is beneficial for a wide range of applications, such as gas separation, surface sensing, and catalysis. In the field of gas separation, the presence of 2-D MOFs in MOF/polymer mixed-matrix membranes will lead to selective transport of preferably adsorbed species in MOFs, such as CO2, but not other gases, resulting in improved selectivity with uncompromised permeance. However, challenges still exist in the facile synthesis of 2-D MOFs due to the isotropic nature of most MOF structure building units. In this work, we demonstrated a one-step room-temperature synthesis of 2-D MOFs based on copper paddlewheel units, where intrinsically anisotropic building units were utilized to control the morphology of MOF nanoparticles. This synthesis method was successfully extended to functionalized 2-D MOFs by selecting ligands with functional groups targeted for CO2 separation applications. To illustrate the advantage of using 2-D MOFs, the synthesized 2-D MOFs were incorporated into polymer matrices and the resultant mixed matrix membranes (MMMs) exhibited improved CO2/CH4separation performance with ideal selectivity increased by approximately 400%. The mechanisms underlying the increased selectivity of MMMs can be attributed to the greatly enhanced sorption toward CO2 molecules as a result of the incorporation of functionalized 2D MOFs, which overwhelmed the impeded gas diffusion owing to reduced chain mobility. Due to the high tunability of MOF structure with retained 2-D morphology, this approach of making 2-D MOFs and its composite materials will be further developed in the separation of other industrially and environmentally important gases.