|ε-Poly-L-lysine (ε-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of Llysine, which is used as a safe food preservative. The present study investigates the combined use of cell immobilization and in situ adsorption (ISA) to produce ε-PL in shaken flasks. Loofah sponge-immobilized Streptomyces ahygroscopicus GIM8 produced slightly more ε-PL than those immobilized on synthetic sponge, and sugarcane bagasse. Moreover, loofah sponge supported the maximum biomass. Hence, loofah sponge was chosen for cell immobilization. Meanwhile, the ion-exchange resin D152 was employed for ISA. The loofah sponge-immobilized cells produced 0.54 ± 0.1 g/l ε-PL, which significantly increased to 3.64 ± 0.32 g/l after combining with ISA through the addition of resin bags. The free cells with ISA using the dispersed resin yielded 2.73 ± 0.26 g/l of ε-PL, an increase from 0.82 ± 0.08 g/l. These data illustrate that the proposed combination method improved production most significantly compared with either immobilization or ISA only. Moreover, the immobilized cells could be repeatedly used and an ε-PL total amount of 8.05 ± 0.84 g/l was obtained. The proposed combination method offers promising perspectives for ε-PL production.