ε-Polylysine (ε-PL) is a highly safe natural food preservative with a broad antimicrobial spectrum, excellent corrosion resistances, and great commercial potentials. In the present work, we evaluated theε-PL adsorption performances of HZB-3B and D155 resins and optimized the adsorption and desorption conditions by single-factor test, response surface method, and orthogonal design. The complexes of resin andε-PL were characterized by SEM and FITR. The results indicated that D155 resin had the bestε-PL adsorption performance and was selected for the separation and purification ofε-PL. The conditions for the static adsorption ofε-PL on D155 resin were optimized as follows:ε-PL solution 40 g/L, pH 8.5, resins 15 g/L, and absorption time 14 h. The adsorption efficiency ofε-PL under the optimal conditions was 96.84%. Theε-PL adsorbed on the D155 resin was easily desorbed with 0.4 mol/L HCl at 30°C in 10 h. The highest desorption efficiency was 97.57% and the overall recovery ofε-PL was 94.49% under the optimal conditions. The excellentε-PL adsorption and desorption properties of D155 resin including high selectivity and adsorption capacity, easy desorption, and high stability make it a good candidate for the isolation ofε-PL from fermentation broths.