A virulent phage P65, one of whose hosts is methicillin-resistant Staphylococcus aureus (MRSA), was isolated from the sewage of dairy cow farm. The transmission electron microscopic (TEM) observation showed that the head of the phage was polyhedral, belonging to the long-tailed Siphoviridae family. Its optimal multiplicity of infection (MOI) was 0.01. The one-step growth curve showed that the latent period of the phage was 10 min, the lysis period was 90 min, and the burst size was 35.46 PFU/cell. Its lytic activity remained stable at pH 4–10 and 30–45 ℃. After 70 min of UV irradiation, the titer of the phage decreased from 1.91 × 10⁸ to 2.65 × 10⁴ PFU/mL. After 48 hours of phage treatment, the biofilm clearance rates of strains MRSA 2 and MRSA 24 were 91.3% and 92.2%, respectively. At 25 ℃, P65 had an antibacterial effect in milk and beef contamination models. The results showed that phage P65 has a wide host spectrum and stable lytic activity, and thus has potential for the removal of biofilm and food preservation.