Lyase, a peptidoglycan hydrolase derived from phage, has been considered as a promising alternative antimicrobial agent. To date, adequate information regarding the characteristics of the Lactobacillus phage lyase is lacking. In this study, a lyase from Lactobacillus phage LFP01 was cloned and heterologously expressed in Escherichia coli (E. coli) for subsequent characterization of the antibacterial activity. The removal efficacy of bacterial biofilm and antimicrobial activity in raw milk were also evaluated. The results showed that LysLFP01 demonstrated broad-spectrum antibacterial activity, surpassing its phage counterpart, with particular efficacy against gram-positive bacteria. It exhibited strong thermostability (4-72 °C) and retained activity across a pH range of 3.0-9.0, although its activity decreased with higher NaCl concentrations. LysLFP01 effectively inhibited and removed Staphylococcus aureus (S. aureus) biofilms, as observed through scanning electron microscopy. Additionally, it exhibited significant antibacterial activity in raw milk at 4 °C, reducing bacterial counts effectively over time. Taken together, these findings indicated the potential of LysLFP01 as a novel and robust antimicrobial agent for food safety applications, particularly in combating S. aureus contamination in low-salt, non-acidic environments.