ε-Polylysine (ε-PL) is a natural and highly effective cationic antimicrobial, of which antibacterial activity is limited in food matrix because of ε-PL's charged amino groups that form complexes with food polyanions. Whey protein-ε-PL complexes delivery system was found to be able to solve the problem and keep the antibacterial activity. This study investigated the antibacterial activity of the complexes and its mechanism against Escherichia coli. The minimal inhibitory concentration of ε-PL was in the range 11.72-25.00 g/mL for the complexes containing different amount of ε-PL and was similar to that of free ε-PL. The results of scanning electron microscopy showed that the complexes could destroy the structure of E. coli cell membrane surface, leaving holes on the surface of the bacteria, leading to the death of the bacteria. The molecular dynamics simulation results showed that the mechanism of the antibacterial activity of the complexes was as follows: under electrostatic interaction, the complexes captured the phospholipid molecules of the bacterial membrane through the hydrogen bonds between the positively charged amino groups of ε-PL and the oxygen atom of the phosphate head groups of the membrane, which could create holes on the surface of the bacteria and lead to the death of the bacteria. The results of activity on real food systems showed that the complexes kept the number of E. coli within 5.8 log10 CFU/g after 7 d storage in sauced duck products, while the positive control (ε-PL) was 6.5 log10 CFU/g and negative control (sterile water) was 7.8 log10 CFU/g. Overall, this study confirmed the antibacterial activity of the complexes and provided fundamental knowledge of its antibacterial activity mechanism.