OBJECTIVES: This study aimed to isolate a phage capable of lysing carbapenem-resistant Klebsiella pneumoniae (CRKP) and to analyse its biological characteristics and whole-genome sequence. METHODS: The phage was isolated and purified from the sewage. Transmission electron microscopy (TEM) was employed to observe the bacteriophage's morphology. Phenotypic characterization of the bacteriophages was determined. The genomic information was analysed. Evolutionary relationships were established through comparative genomics, proteomics, and phylogenetic analysis. RESULTS: The isolation of a virulent phage, named Klebsiella phage vB_KpnM_KpVB3, was notable for forming 6-7 mm transparent circular zones, each surrounded by a distinct halo. The phage had a head diameter of ca. 30 nm and a tail length of ca. 20 nm, being identified as a member of the Myoviridae family and the Caudovirales order. The optimal multiplicity of infection (MOI) was 0.00001, with an incubation period of 20 minutes and a lysis period of 60 minutes, and the number of released phages after lysis was 133±35 PFU/cell. The phage was relatively stable at temperatures ranging from 10°C to 40°C and at pH values ranging from 3 to 11. Its lytic efficiency against CRKP was 30.30%. It has been shown to be able to destroy the biofilm of host bacteria. The bacteriophage genome consists of double-stranded DNA (dsDNA) with a total length of 48,394 base pairs, a GC content of 48.99%, and 78 open reading frames (ORFs). CONCLUSION: The study resulted in the isolation vB_KpnM_KpVB3, a phage demonstrating potential therapeutic efficacy against infections caused by CRKP.