A macroporous and weak acid cation exchange resin, Na+-form Amberlite IRC-50, was employed to separate and purify ε-poly-L-lysine (ε-PL) from fermentation broth. Based on the optimization of uploading, wishing and elution parameters, the optimal dynamic ion exchange conditions were developed as following: the sample was uploaded at pH 7.0 with loading rate at 5 BV/h; deionized water was used for washing resin at 2 BV/h; 0.25 mol/L NaOH was used as eluent with elution rate at 2 BV/h. Under the above conditions, the working exchange capacity of the resin was reached by 305.12 mg/g, and the desorption rate was attained to more than 95%. Finally, the purity of ε-PL was enhanced from the initial 24.29% to 75.26%, and the ε-PL recovery rate was also reached by 96.40%.