Driven by sustainable development strategies, bio-based polyesters have received significant attention due to renewable bio-derived monomers and various unique properties. In this study, a series of high molecular weight bio-based poly(ethylene-co-2,2,4,4-tetramethyl-1,3-cyclobutylene thiophenedicarboxylate) (PETTh) copolyesters on the basis of an emerging bio-derived aromatic diacid, 2,5-thiophenedicarboxylic acid (TDCA), was synthesized by a conventional two-step melt polycondensation method. Copolymers were random copolyesters with weight-average molecular weights (Mw) in the range from 65,400 to 84,100 g/mol. Their comprehensive properties, including thermal transition properties, thermal stability, mechanical properties, gas barrier properties, as well as optical transparency were systematically investigated. Rigid 2,2,4,4-tetramethyl-1,3-cyclobutanediol (CBDO) cyclic diol contributed a positive effect to thermal resistance, leading to the glass transition temperature (Tg) growing from 65.3 °C of PETh to 85.3 °C of PET42Th. Furthermore, the mechanical properties, gas barrier properties and optical transparency were improved as diol segment was incorporated. Especially, all copolyesters displayed great ductility with elongation at break between 54 and 195 %. Meanwhile, the gas barrier properties of copolyesters were 3.9-fold to 16.3-fold for CO2 and 3.0-fold to 6.0-fold for O2 better than PET. Therefore, PETTh copolyesters might potentially substitute for commercial PET and they are extremely suitable for food packaging materials.