A combination treatment of ultrasound and ε-polylysine to improve microorganisms and storage quality of fresh-cut lettuce were investigated. Fresh-cut lettuce was treated with ultrasound (20 kHz, 17–29 W/L), ε-polylysine (0.1–0.6 g/L) and their combination at 20 °C for 10 min and then packaged as well as stored at 4 °C for 12 d. The results showed that 23 W/L ultrasound combined with 0.4 g/L ε-polylysine treatment was superior to 23 W/L ultrasound or 0.4 g/L ε-polylysine treatment alone in inhibiting the growth of microorganisms such as total number of colonies, mold and yeast, and total coliform counts of fresh-cut lettuce during storage. Ultrasound combined with ε-polylysine treatment reduced weight loss and total color difference (ΔE), declined POD and PPO activities, maintained the higher level of total phenolics content, vitamin C and chlorophyll, as well as decreased water mobility and respiration rate in fresh-cut lettuce during storage compared to control. Therefore, these results demonstrated that ultrasound combined with ε-polylysine treatment was helpful for inhibiting microorganisms and improving storage quality, and can be an effective preservation method to extend shelf-life of fresh-cut lettuce.