Abstract: An experimental apparatus for separation of hydrogen isotopes was developed by using multi-bed palladium displacement chromatography. The effects of vacuum treatment time of palladium column, extraction proportion and the composition of original gas on separation efficiency were also studied. The results indicate that with more than three hours vacuum treatment for the separation column at 500-550℃ and keeping the temperature rise rate at 10℃/min, high pure deuterium or tritium can be obtained. As for a mixture of protium and deuterium gas, less than 1.0%(volume fraction) protium can be obtained under appropriate proportional extraction.