The gas separation performance of mixed-matrix membrane (MMM) highly depends on the properties of nano-fillers and polymer matrix and their interfacial compatibility. Herein, we report the fabrication of a COF-based MMM (COF-300-Amide/Pebax2533) for CO2 separation, in which highly crystalline amide-linked COF-300-Amide particles used as nano-additives and incorporated into Pebax2533 polymer via solution casting method to obtain the membrane. The COF-300-Amide was obtained by oxidizing imine-linked COFs (COF-300) for irreversible and high crystalline synthesis. The membrane of COF-300-Amide/Pebax2533 showed uniform and continuous morphology, which demonstrated that the nano-particles had good interfacial compatibility with the Pebax2533 matrix. GCMC simulation and dcTST method were used to calculate the CO2 adsorption/diffusion properties in COFs. The interfacial interaction between the nano-fillers and substrate was thoroughly quantified by molecular dynamics (MD) simulations. The resultant membrane demonstrates a simultaneous enhancement of individual gas permeability (681.6 Barrer) and CO2/N2 ideal selectivity (41.3), surpassing Robenson upper bound (2008). Moreover, the synthesized COF with high crystallinity greatly enhanced the stability of COF-300-Amide/Pebax2533 with an anti-decomposition temperature of 397.1 °C and a lasting sustainability over 28 days. The study demonstrates the great potential of the developed COFs-based MMMs in promoting CO2 separation applications.