Lactococcus lactis, a candidate probiotic, is extensively applied in the fermentation of the food industry and is recognized for its potential to enhance human health. In this study, L. lactis ZFM559, which exhibits probiotic properties, was isolated from fresh milk. In vitro assays have demonstrated it has the ability to inhibit and co-aggregate with multiple pathogenic strains, as well as good tolerance to high osmotic pressure (10% NaCl), low pH (3.0), and simulated gastrointestinal conditions. Moreover, L. lactis ZFM559 is capable of producing folate and inhibiting α-glucosidase activity. Whole-genome sequencing has revealed the components of its genome, which include one circular chromosome and three linear plasmids. Functional annotation of the genome has identified the pathway for γ-aminobutyric acid synthesis, genes and enzymes associated with flavor production and lactose decomposition, and proteins related to environmental stress tolerance. Additionally, a complete gene cluster (including LanT, HlyD, LcnB, and GlyS) for the synthesis of bacteriocins was identified. In summary, L. lactis ZFM559 can be considered a probiotic with significant potential for applications in the food industry and human health.