Streptomyces albulus TUST2, separated from a soil sample of Hainan Island, was cultivated in a stirred, submerged aerated 5-1 bioreactor. Carbon source and nitrogen source effects were clarified in fed-batch fermentation, particularly glucose and ammonium sulfate feedings for enhancing the epsiv-poly-L-lysine (epsiv-PL) production were elucidated. The culture was divided into two control phases. In phase I , the pH was maintained at its initial 6.8 for 36 h for acquiring high biomass; In phase II, the pH was maintained at 4.0 aiming at increasing the epsiv-PL accumulation. Moreover, pulsed feeding of glucose (800 g/l) was carried out accompanied by ammonium sulfate (80 g/l) discontinuously when their concentration reached a lower level (nearly 10 g/l) in the medium, so that the final concentration of glucose can be kept at 13 g/l. By using this feeding policy, the elevation of antibacterial activity of epsiv-PL by S. albulus TUST2 is 16.67%, and its accumulation in 4-days cultivation is 20.20 g/l. We have illuminated that substrate feeding strategy is a doable method for improving epsiv-PL production by S. albulus TUST2.