Abstract: A highly efficient separation method for trace plutonium from high uranium plutonium ratio samples was established by combining two Reillex anion exchange chromatographic columns. The effects of resin type, wash condition, valence state adjustment on plutonium separation were investigated, and the conditions of separation process were determined. When the mass ratio of uranium to plutonium in the feed liquid is 106, after two-stage Reillex column separation, the average yield of plutonium is 99.35% and the decontamination factor of uranium is greater than 10⁷. Using this process, high purity trace ²³⁹Pu is separated from irradiated uranium target, and the atomic ratio of (²³⁸U+²³⁸Pu)/²³⁹Pu is (3.60±1.15)×10^-5(n=6).