Abstract: Three kinds of graphene oxide (GO) with different oxidation degrees were synthesized by Hummers method. The GO/polyurethane (PU) mixed matrix membranes were fabricated via in-situ polymerization of PU monomers (Methylene Diphenyl 4, 4'-Diisocyanate (MDI) and 1, 4-Butanediol (BDO)) with GO. The structure of GO was characterized by XRD, Raman, FTIR and TEM etc. The effects of GO filling content on the morphology and permeabilities of CO₂ and N₂ of GO/PU mixed matrix membranes were discussed. The results reveal that the three kinds of GO with different oxidation degrees present a state of complete stripping, and are translucent sheet structure, and with the increasing of oxidation degree, the relative intensities of D peak to G peak in Raman are 0.947, 1.103 and 1.245. The oxidation degree of GO has greater effects on the dispersibility of GO in solution and mixed matrix membrane, when the oxidation degree is higher, the dispersibility is better. The permeability coeffcients of CO₂ and N₂ and CO₂/N₂ permeation selectivity factor of GO/PU mixed matrix membrane increase firstly and then decrease with GO filling content increasing. When the mass ratio of middle oxidation degree GO (M-GO) to (MDI+BDO) reaches 1.0%, the CO₂ permeability coefficient of M-GO/PU mixed matrix membrane is 63.6×10^-13 cm³(STP)/(cm·Pa·s), STP represents for standard temperature and pressure, and the permeation selectivity factor of CO₂/N₂ reaches 48.5. The filling of appropriate amount of GO could significantly improve the permeability of CO₂ and the CO₂/N₂ permeation selectivity of GO/PU mixed matrix membrane.