Electrolysis of water is widely used for hydrogen isotope separation and the development of hydrogen evolution reaction (HER) catalysts with high selectivity and activity is of key importance. Herein, we propose single atom catalysts (SACs) as promising catalysts for efficient hydrogen isotope separation. Pt SACs and Pt nanoparticles (NPs) have been fabricated on nanoarray-structured nitrogen-doped graphite foil (NGF) substrate by a polyol reduction method. The as prepared Pt₁/NGF electrode exhibits high activity and selectivity toward HER with a low overpotential of 0.022 V at 10 mA·cm⁻² and a high separation factor of 6.83 for hydrogen and deuterium separation, much better than Pt NPs counterpart. Density functional theory (DFT) calculations ascribe the high activity and selectivity to the constructed Pt-N₂C₂ structure. This work develops a new opportunity for the design and application of high-efficiency and stable SACs toward hydrogen isotope separation by electrolysis of water.