Metal-organic framework (MOF) nanosheets have been identified as promising membrane materials for various separation applications. However, the development of MOF nanosheet membranes for practical applications is impeded by the difficulties in preparing ultrathin nanosheets and the limitations of conventional membrane production methods. For the first time, conveniently prepared and purified isoreticular MOF-3 (IRMOF-3) nanosheets are filled into macroporous glass fiber (GF) filters to prepare IRMOF-3 nanosheet-filled GF (IRMOF-3@GFX, X = filling amount expressed in mg) membranes. The IRMOF-3@GF2.0 membrane exhibits an impressively high H2/CO2 selectivity of 1341 ± 134, and the H2 permeance reaches 974 ± 41 gas permeance units (GPU). In addition, the IRMOF-3@GF2.0 membrane shows excellent durability with no performance degradation during a 150-h test. Grand Canonical Monte Carlo simulations are performed to investigate the H2/CO2 separation mechanism and the results suggest a synergistic separation mode based on selective adsorption and size exclusion. The innovative filled-membrane fabrication route and the presented gas separation mechanism offer a new strategy to design and prepare MOF nanosheet-based gas separation membranes.