The effects of concentration of extractant (0.05 mol/L, 0.1 mol/L, 0.15 mol/L), pH (3.08, 3.14, 3.2) and the flow rate of mobile phase (0.25 mL/min, 0.5 mol/L, 0.75 mol/L) on the separation efficiency of Am and Eu by high speed countercurrent chromatography (HSCCC) were investigated using dichlorophenyldithiophosphinic acid in xylene as the stationary phase and 0.1 mol/L NaClO4 as mobile phase. The results show that the separation factor increases with the increasing of the concentration of extractant and the pH value of the mobile phase, furthermore, minishing the flow rate of the mobile phase can also improves the separation efficiency between Am and Eu. Mutual separation between Am3+ and Eu3+ can be accomplished Eu3+ by optimizing the separation condition, the separation factor and separation efficiency between Am3+ and Eu3+ can reach 2.87 and 0.74, respectively.