By mixing functionalized carbon nanofibers as inorganic substances with polysulfone (PSF) membrane, an extraordinary ability to separate CO2 from CH4 as well as N2 from O2 was developed in this study. The series of amine-functionalized-CNFs (F-CNFs)/PSf mixed matrix membrane with various F-CNFs contents (0–1.5wt.%) was prepared by solution casting method. In this paper, characterizations such as; morphology and crystalline structure have been conducted in details to investigate the structures and properties of mixed matrix membranes. The effects of F-CNF loading on the gas separation (O2, N2, CO2 and CH4) performance of the MMMs were evaluated using laboratory-scale gas separation apparatus. The MMMs exhibited higher CO2 permeability and perm-selectivity of CO2/CH4 due to higher interaction of amine groups on F-CNFs with CO2 as compared with the neat PSf membrane. The permeation measurement showed the CO2 and O2 permeability of about 3.57 and 1.58 (GPU), and CO2/CH4 and O2/N2 selectivity of about 7.7, 3.5, respectively, were obtained for MMM with 0.5wt.% F-CNFs loading. As a result, the developed membranes can be efficient for separating CO2 from CH4. The N2 selectivity was increased with addition of 0.5wt.% F-CNFs in the PSf casting solution, and then decreased with higher loading.