〔Abstract〕 To isolate a lytic phage targeting K. pneumoniae type K39 from untreated sewage and systematically analyze its biological characteristics and genomic information. Methods The bacteriophage ofK. pneumoniae K39 Kp1000 was identified based on the sequence of the capsular polysaccharide gene. It was isolated and purified using the double agar plate method. The morphology of the phage was observed through negative staining and transmission electron microscopy, and its bacteriophage spectrum was evaluated by phagocytosis. The biological characteristics of the bacteriophage were assessed by determining the optimal multiplicity of infection (MOI), constructing a one-step growth curve, and conducting temperature and pH tolerance tests. Finally, the phage DNA was extracted, and its whole genome was sequenced using the Illumina HiSeq2000 sequencing platform. The sequencing results were then annotated and analyzed. Results A lytic phage specifically targeting K. pneumoniae type K39 was successfully isolated and named IME330. Transmission electron microscopy showed that the head diameter of the phage was (75±1) nm and the tail length was (185±1) nm. The lytic spectrum analysis revealed that IME330 lysed K. pneumoniae type K39, demonstrating a narrow host range and high specificity. The optimal multiplicity of infection (MOI) was 0.1, The lysis amount reached 168 PFU/cell. Physicochemical experiments indicated that IME330 exhibited strong tolerance to high temperatures and a broad pH range (pH 4-10). Genomic analysis demonstrated that the IME330 genome had a length of 144,245 bp, a molecular weight of 46,463 MDa, a G+C content of 44.8%, and encoded 320 open reading frames (ORFs). Conclusion IME330 is a novel lytic K. pneumoniae phage belonging to the order Caudovirales. This phage exhibits strong tolerance to physicochemical factors (e.g., temperature, acidic/alkaline conditions) and demonstrates high lytic activity, while its lytic spectrum remains narrow with strict host specificity.