As a common foodborne pathogen, Listeria monocytogenes is a major threat to the safety of fruit juice. In this study, The activity and action mechanism of antimicrobial peptide zp37 in inhibiting L. monocytogenes in fruit juice were studied by measuring its minimum inhibitory concentration (MIC), and analyzing its growth curve, membrane potential, membrane integrity, cell microstructure, cell aggregation rate, DNA binding, intracellular reactive oxygen species (ROS) level after zp37 treatment. The results showed that the MIC value of antimicrobial peptide zp37 against L. monocytogenes was 16 μg/mL, and the growth of L. monocytogenes was significantly inhibited by zp37 at concentrations greater than 4 MIC. After treatment with the peptide at 8 MIC concentration for 1 h, L. monocytogenes was almost completely killed. The peptide at 2 MIC concentration kept the number of L. monocytogenes in strawberry, kiwifruit and apple juice below the detection limit for seven days. After treatment with zp37, the cell membrane permeability of L. monocytogeneswas increased, the membrane potential was lost, the cell membrane integrity was destroyed, the cell surface became obviously sunken and deformed, and cell aggregation occurred. The results of fluorescent labeling showed that zp37 could simultaneously act on the cell membrane and cytoplasm of L. monocytogenes. In the cytoplasm, when zp37 concentration was greater than one-fourth of the DNA concentration, they could bind significantly and even DNA was precipitated. In addition, zp37 induced L. monocytogenes to produce ROS in a positive dose-dependent manner. The above results showed that antimicrobial peptide zp37 had anobvious inhibitory effect on L. monocytogenes in fruit juice, which can provide a scientific basis for the application of antimicrobial peptides as biological preservatives in fruit juice.