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Carbon dioxide (CO2) is considered one of the causes of global warming because anthropogenic CO2 concentration in the atmosphere has greatly increased. The objective of this work was to design silk fibroin (SF) and graphene oxide (GO) membranes with very high CO2 gas capture characteristics and high gas selectivity toward nitrogen rather than CO2 gas, and to demonstrate the effect on transforming the SF secondary structure at the molecular level. GO-NH3+/SF multilayer nanocoatings were fabricated by automatic spray-assisted layer-by-layer (LbL) assembly with physical molecular interaction between GO-NH3+ and SF. We demonstrated transformation of the SF secondary structures in GO-NH3+/SF LbL-assembled nanocoatings with MeOH solvent treatment. After MeOH treatment, the SF secondary structures in the GO-NH3+/SF LbL-assembled nanocoatings preferred beta-sheets to random coils. This phenomenon affects the internal amine groups and CO2 transport. The MeOH-treated GO-NH3+/SF LbL-assembled nanocoatings have high beta-sheet content, and the gas permeability of the nanocoatings was quite different. Before the predominance of the beta-sheet, CO2 gas permeated twice as fast as nitrogen (N-2) gas in the GO-NH3+/SF LbL-assembled nanocoatings. On the other hand, CO2 gas was transmitted 10-times slower than N-2 gas in MeOH-treated GO-NH3+/SF LbL-assembled nanocoatings. In other words, we successfully fabricated a CO2 adsorptive separation membrane with GO-NH3+ and SF.