Abstract: Enhancing the vitality of probiotics in probiotic product while inhibiting the growth of food-borne pathogenic and spoilage bacteria can improve product quality stability. Under mono-culture and co-culture conditions, the effects of different concentrations of tea polyphenols on the growth of Lactobacillus plantarum, Staphylococcus aureus, and Escherichia coli were comparatively investigated by culture-dependent method and high-throughput sequencing. Mono-culture result showed that with the increase of tea polyphenols concentration, the viable count of L. plantarum first ascended and then decreased, reaching the maximum at the concentration of 2.0 mg/mL. However, the survival rate of the two pathogens decreased continuously, with S. aureus decreasing more significantly. In the co-culture system (S. aureus/E. coli-L. plantarum), the biomass of L. plantarum showed a significant increase with culture time, coupled with a general decrease in the number of the pathogen and pH of the medium. In addition, Illumina Miseq sequencing further showed that the relative abundance of Lactobacillus was higher than that of the control when coexisted with the pathogen in the presence of tea polyphenols, while the relative abundance of the pathogen was lower than that of the control. The results indicated that tea polyphenols at an appropriate concentration (2~4 mg/mL) had a dual-directional regulation on the growth of L. plantarum and pathogens, that was, proliferating L. plantarum while inhibiting the growth of pathogens.