Epsilon-poly-lysine (EPL), a natural cationic antimicrobial peptide, is widely used in food processing and preservation. However, when applied to fruit and vegetable juice, EPL can cause turbidity and precipitation, which reduces the antimicrobial properties and juice quality. As a colloidal delivery system, nanoliposome provides a novel insight into improve the activity of EPL. In this study, the antibacterial activity and mechanism of chitosan-coated EPL nanoliposomes (CS-EPLLP) against Alicyclobacillus spp. in apple juice were evaluated. It was found that the minimum inhibitory concentration of CS-EPLLP against the strains of A. acidocaldarius, A. fructus and A. acidoterrestris were in the ranges of 0.625∼1.25 mg/L, while the minimum bactericidal concentration of CS-EPLLP against these strains in apple juice were 0.625-2.5 mg/L. Compared to A. fructus and A. acidoterrestris, A. acidocaldarius displayed a better resistance to CS-EPLLP. Meanwhile, the modification of chitosan can improve the bactericidal effect of EPL and EPL nanoliposomes. In addition, CS-EPLLP treatment can lead to the inactivation of bacteria cells by causing surface damage, destroying their membrane integrity, preventing the intracellular ATP synthesis, increasing intracellular protein leakage and cells membrane permeability. Furthermore, the addition of CS-EPLLP in the amounts of MBC had no significant effect on the color change, transmittance, centrifuge sedimentation rate and main volatile compounds of apple juice. The prepared CS-EPLLP and established method provides a new insight into the inactivation of bacteria in fruit juice.