For decontamination and quantification of trace amount of tritium in water, an efficient separation technology capable of enriching tritium in water is required. Electrolysis is a key technology for tritium enichment as it has a high H/T and D/T separation factors. To separate tritium, it is important to develop a proton exchange membrane (PEM) electrolyzer having high hydrogen isotope separation factor as well as high electrolyzer cell efficiency. However, there has not been sufficient research on the separation factor and cell efficiency according to the composition and manufacturing method of the membrane electrode assembly (MEA) Therefore, it is necessary to study the optimal composition and manufacturing method of the MEA in PEM electrolyzer. In this study, the H/D separation factor and water electrolysis cell efficiency of PEM electrolyzer were analyzed by changing the anode and cathode materials and electrode deposition method of the MEA. After the water electrolysis experiment using deionized water, the D/H ratio in water and hydrogen gas was measured using a cavity ring down spectrometer and a mass spectrometer, respectively, and the separation factor was calculated. To calculate the cell efficiency of water electrolysis, a polarization curves were obtained by measuring the voltage changes while increasing the current density. As a result of the study, the water electrolyzer cell efficiency of the MEA fabricated with different anode/cathode configurations and electrode formation methods was higher than that of commercial MEA. On the other hand, the difference in H/D separation factor was not significant depending on the MEA fabrication methods. Therefore, using a cell with high cell efficiency when the separation factor is the same will help construct a more efficient water electrolysis system by lowering the voltage required for water electrolysis.