A bacteriophage of E.coli SPHJ965 was isolated from domestic sewage and named as ΦSPHJ965. The stability of the bacteriophage to temperature and pH, the sensitivity to UV and chloroform were further analyzed. The optimal multiplicity of infection and one-step growth curve were determined. Finally, the preliminary application of E.coli removal was carried out. The results showed that the titer of ΦSPHJ965 was 3.4×10⁹ PFU/mL, and it had strong tolerance to pH and chloroform, but it was sensitive to temperature and UV. The titer decreased significantly when incubated above 60 ℃. After UV irradiation for 30 min, it lost its lytic ability to host bacteria. The optimal multiplicity of infection was 0.00001, The phage demonstrated the incubation period was 10 min, burst period was 90 min and burst amount was 40 PFU/cell. The antibacterial experiment showed that ΦSPHJ965 had a good inhibitory effect on the host bacteria within 6 h. After 6 h, the OD₅₉₅ value of the experimental group increased gradually, but remained far lower than that of the control group, indicating that the phage had a good lytic effect on the host bacteria, but it could not completely lyse. After the wastewater was treated with ΦSPHJ965 for 1 h, the total number of colonies and Escherichia coli decreased significantly, indicating that the phage had good antibacterial effect. In conclusion, ΦSPHJ965 has high titer, strong tolerance to temperature and pH, and good removal effect on Escherichia coli, which has potential application as a biological scavenger of Escherichia coli.[Chinese Fishery Quality and Standards,2023,13(5):31-38]