6FDA-polyimide films modified by polyamidoamine (PAMAM) dendrimers with generations of 0, 1, and 2 are reported in this article. The actual molecular conformation and bulk size of these three generation dendrimers immobilized on polyimide surface were characterized by atomic force microscopy. After comparing with the results of dynamic simulation, we believe that the disk-shape cluster structure of dendrimers has been developed on the polymer surfaces. The amidation and cross-linking reaction between dendrimers and polyimide were examined and quantified by X-ray photoelectron spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and gel content measurements. Modification time and the generations of PAMAM dendrimer have been verified as two important factors in determining the properties of modified polyimide films. These modified polyimide films exhibit excellent gas separation performance. The ideal selectivity of He/N-2 increases tremendously to about 200% as compared to that of the original polyimide film. Particularly, the separation performance of CO2/CH4 gas pair can be improved beyond the upper bond limit possibly due to the strong interactions of dendrimer molecules with CO2, which was verified by sorption tests.