It’s necessary to establish a precision analytical method of Gas Chromatography (GC) for achieving the detection of isotopes safely and effectively in the fuel cycle system of fusion reactors. The studies have shown that the separation of H2, HD and D2 in high purity helium with handmade cryogenic packed separation column of Al2O3, which was coated with the liquor of MnCl2 and finally measured by Discharge Ionization Detector (DID). This paper investigated that with the carrier gas flow rate ranging from 4.0 to 25.0 mL/min, the separation of isotopes with the optimum resolution of H2-HD and HD-D2 were 1.067 and 1.632, respectively, which exceeded 1.0 and could achieve effective separation with high column efficiency and good repeatability. Such a detection technology of H2, HD and D2 could be applied to Fuel systems (FS) and Isotope Separation System (ISS) in Tritium Plant of inner fuel cycle, for further improvement of separating H2, HT and T2 could be applied to Tritium Extraction System (TES) and Coolant Purification System (CPS) in Test Blanket Module (TBM) of fusion reactors, so as to ensure the accountancy of tritium safely and effectively.