Abstract: In this study, an on-line Gas Chromatography-Pyrolysis-Gas Chromatography-Isotope Ratio Mass Spectrometer (GC-Py-GC-IRMS) was established to conduct position-specific isotope analysis (PSIA) by enrichment of compound interested, chromatographic separation, instantaneous pyrolysis and isotope ratio measurement. Propane, as its instantaneous pyrolysis can be kinetically controlled, was selected for tests. The molar conversion (mol%) of propane during pyrolysis and the carbon isotopic compositions of pyrolysis products upon temperature sequence show that the optimal pyrolysis temperature of propane is 780-820 ℃ for its position-specific carbon isotope analysis. Integrated with carbon isotopic fractionation during the propane pyrolysis, the carbon isotopes of central and terminal carbon were successfully calculated. Two natural gas samples from the Daniudi Gas Field, Ordos Basin were collected for central and terminal carbon isotope measurements in propane. Similar δ¹³C values of central carbon of propane in natural gas from the Ordovician and Carboniferous-Permian reservoirs could be indicative of the same source strata. While ¹³C-enrichment in the terminal C-atom of propane in natural gas from the Carboniferous-Permian reservoirs probably indicates that natural gas accumulated in the Carboniferous-Permian reservoir maybe have experienced a higher thermal maturation compared to that from the Ordovician reservoirs. The results suggest that the PSIA in propane can be a potentially powerful tool to probe the mechanisms on natural gas generation.