Quality deterioration and aroma loss of vacuum-packed fresh-cut lettuce during storage are important factors affecting its commercial value. In this study, the effects of ε-polylysine (ε-PL) on the quality and flavor attributes of vacuum-packed fresh-cut lettuce were studied. Fresh-cut lettuce was treated with different concentrations of ε-PL (0.2, 0.8, and 1.5 g L−1), vacuum-packed and stored at 4 °C for 12 d. The results showed that ε-PL treatment maintained the color and texture of fresh-cut lettuce, and reduced chlorophyll degradation and ascorbic acid loss. In addition, ε-PL treatment inhibited the growth of aerobic bacteria, and maintained or increased some aroma components such as (E)-2-hexenal, n-hexanol, hexanal, limonene, benzaldehyde. These results indicate that ε-PL treatment has potential practical application value in the preservation of vacuum-packed fresh-cut lettuce.