[Background] Phage can specifically kill host bacteria especially the strains resistant to antibiotics. Phage can be a new antimicrobial agents alternative to conventional drugs. [Objective] No Enterobacter ludwigii phage has been reported until now. The purpose of this work is to isolate and characterize E. ludwigii phage. [Methods] The double-layer plate method was used for isolation, purification, and quantification of phage; SDS-PAGE was used for viral structural protein analysis; transmission electron microscopy (TEM) was used for phage morphology analysis; and the Crystal Violet staining and Congo Red agar methods were used for biofilm analysis. [Results] The phage GM20 was isolated from the environmental samples with the strain E. ludwigii GM20 as indicator. The plaques were transparent with the average diameter about 0.47 mm. TEM showed that GM20 has a contractile tail, and it was classified as a member of the Myoviridae family. The titer of phage is 7.65′109 PFU/mL, suggesting that it is a virulent phage. The one-step growth curve showed that GM20 had a latent period of 15 min and a burst size of 164.3 PFU/infection center. Further experiments showed that GM20 could inhibit bacteria growth effectively, and the average mutation frequency of phage-resistant mutants was about 1.06′10?5. [Conclusion] The virulent phage GM20 can effectively kill the host strain E. ludwigii X20 and may be used in the prevention and control of E. ludwigii infection.