[Background] With the increasingly serious drug resistance of Enterococcus faecium, it is urgent to screen and obtain new lytic bacteriophages with high lysis efficiency and clear genetic background, to enrich phage resources and provide appropriate strains for phage therapy. [Objective] To evaluate the biological characteristics and genome analysis of an Enterococcus faecium bacteriophage isolated from the fecal sample of Nanjing Xigang Dairy Farm. [Methods] The bacteriophage was purified by double-layer agar plate. The purified bacteriophage was negatively stained with phosphotungstic acid and observed by the transmission electron microscope (TEM). Then, one-step growth curve, pH tolerance, temperature tolerance, and optimal multiplicity of infection were detected. Also, its genomics characteristics and evolutionary history were analyzed. [Results] The results are as follows:A lytic Enterococcus faecium bacteriophage, named as vB_EfaS_29, was isolated and purified. Its plaques were round, bright, and clear. TEM analysis revealed that bacteriophage vB_EfaS_29 had a twenty-side head and a long tail. The head diameter was about 52.4 nm, and the tail length was about 157.1 nm. It belongs to the Siphoviridae family. The optimal multiplicity of infection of bacteriophage vB_EfaS_29 was 0.1; the maximum tolerable temperature was about 70℃; during pH 6.0-9.0, it was stable. The complete genome sequences of bacteriophage vB_EfaS_29 comprised of 41 014 bp with a GC content of 34.99% and contained 63 putative open reading frame (ORF). Only 36 of the ORF were well predicted for functions. Undesirable genes such as lysogenic, antibiotic resistance, or virulence were not detected in the genome. The phylogenetic analysis of vB_EfaS_29 based on amino acid of the endolysin, demonstrated that vB_EfaS_29 had more than 99% of sequence homology to FelixO1-like phages. However, using the amino acid of capsid as the targeted protein, vB_EfaS_29 had a long genetic distance with other Enterococcuss bacteriophages. [Conclusion] The above results provided theoretical data and resources for bacteriophage research and bacteriophage therapy.