In this paper, a new chemistry approach was developed for synthesis of castor oil (CO)-based polyurethane (PU) membranes to attain improved structural properties for gas separation applications. For this purpose, propyne-terminated CO-based PU prepolymer (PTPU) was synthesized by the reaction of CO and isophorone diisocyanate (IPDI), followed by propargyl alcohol (PrAl) and isocyanate (NCO) terminated prepolymer reaction. The ultimate membranes were prepared through thiol-yne cross-linking reaction of PTPU and pentaerythritol tetrakis (3-mercaptopropionate) (PETMP), in presence of azobisisobutyronitrile (AIBN), as a reaction initiator. It was revealed that mechanical and thermal properties of the prepared membranes were improved owing to the formation of flexible thioether linkages through cross-linking reaction. The gas permeation measurements were carried out in the temperature and pressure ranges of 298–338 K and 200–1200 kPa, respectively. The results exhibited reverse size selective performance, as a typical transport behavior of rubbery membranes. The infinite dilution permeability coefficient of CO2 at 298 K was found identical to 2.78 Barrer, along with the infinite dilution perm-selectivity values for CO2/CH4 and CO2/H2 obtained equal to 25.27 and 7.94, respectively. Furthermore, higher permeation activation energies were found in this work compared to a number of cross-linked rubbery membranes in the literature, such as poly (dimethylsiloxane) (PDMS) and poly(ethylene glycol) diacrylate (PEGDA).