Carbon molecular sieve (CMS) membranes have been proven to be effective gas separation membranes, especially for light gas separation. In the current work, Troger's base polymer (TB)/poly(styrene sulfonic acid) (PSS) thin-film-composite (TFC) carbon molecular sieve (CMS) membranes were prepared by dip-coating and subsequent carbonization. Porous anodic aluminum oxide (AAO) plates were employed as the support layer, and carboxylated nanocellulose fiber (CNF) was used as the sacrificed layer. The effects of membrane preparation conditions, including solution concentration and carbonization temperature, and operating conditions, such as feed pressure and test temperature, on the hydrogen (H 2 ) and helium (He) separation performance were systematically investigated. Meanwhile, the relationship between gas separation performances and the microstructure of TFC CMS membranes was explored by means of scanning electron microscopy (SEM), X-ray diffraction (XRD), and Raman spectrum. With a carbonization temperature of 550 °C, the H 2 /CH 4 (149.25), H 2 /N 2 (131.04), He/CH 4 (137.07), and He/N 2 (120.35) selectivity of the TB/PSS membrane prepared at a solution concentration of 10 wt % was significantly higher than other TFC CMS membranes, and the H 2 and He permeances were 1359.73 GPU and 1248.49 GPU under a feed pressure of 2 bar, respectively. The excellent gas separation performance of this membrane demonstrates its great potential for H 2 and He purification applications.