epsilon-Poly-L-lysine (epsilon-PL) serves as a biological preservative in food industry for many years in several countries. However, the naturally occurring epsilon-PL with the chain length of 25-35 L-lysine residuals exhibits bitter taste. Thus, the decrease of chain length at an appropriate range has become very critical for the wide application of epsilon-PL. Herein, we proposed an efficient strategy for the short chain epsilon-PL production with the high yield (39.84 g/L), high purity (98.81%) and high recovery ratio (72.59%) by fed-batch fermentation using glycerol as carbon source. The short chain epsilon-PL with 8-32 L-lysine residuals showed different secondary structures and better antimicrobial activity towards yeast than naturally occurring one. As a result, a simple, low-cost and safe strategy for high-level production of short chain epsilon-PL was developed, which may enlarge the application of epsilon-PL in the food industry.