The MOF Ni(bdc)(ted)0.5 was selected as the hydrogen isotope separation material in this paper and the separation mechanism of this material was investigated by molecular simulation. Theoretical calculations proved that Ni(bdc)(ted)0.5 had chemical affinity quantum sieving (CAQS) effect and kinetic quantum sieving (KQS) effect. Because the adsorption capacity of D2 in Ni(bdc)(ted)0.5 was larger than H2 and the diffusion rate was smaller than H2 at 77 K, Ni(bdc)(ted)0.5 could realize the separation of hydrogen and deuterium in gas chromatography. Ni(bdc)(ted)0.5@γ‐Al2O3 composite was prepared by liquid phase epitaxy method as gas chromatographic packings (column type: 0.6 m×2.0 mm I.D.) to investigate the separation performance of H2/D2 at 77 K. Under the optimal chromatographic conditions, the retention time of H2 and D2 were 1.95 and 3.48 min respectively and the resolution (R) was 2.12, indicating that the separation of H2 and D2 could be realized efficiently and quickly by using Ni(bdc)(ted)0.5@γ‐Al2O3.