Abstract: Separation of nickel and cobalt from bioleaching solution was studied using a synergistic extraction system of P507 and Cyanex272. Test results indicate that the system has a better extraction effect. Combined with the system characteristic of high acidity and low cobalt-to-nickel ratio, the separation effect of nickel and cobalt was compared in three extraction systems of P507, Cyanex272, and P507-Cyanex272. P507-Cyanex272 was identified to have a good separation effect of nickel and cobalt at the initial pH value of 1.5 to 2.2 and the balance pH value of 4.00 to 5.25. Influence factors on separation of nickel and cobalt in the extraction system at 28℃ were examined systematically, and the best processes for this synergistic extraction are the P507/Cyanex272 mole ratio of 3:2, the saponification rate of 60%, the extractant volume fraction of 10%, and the organic to aqueous phase ratio of 1:4. Under these conditions, the first extraction rate of cobalt is 99.16%, and the separation factor of nickel and cobalt is 932.59.