Abstract: OBJECTIVE To analyze the biological characteristics and whole-genome features of the multidrug-resistant Pseudomonas aeruginosa phage YN02 so as to provide theoretical bases for prevention and control of infections and clinical application of phages. METHODS The strains that were isolated from sputum samples of the tracheotomy patients with postoperative pulmonary infections were identified, the antimicrobial susceptibility testing was performed, the strains were treated as host bacteria, and the phages were isolated from contaminated water of the hospital. Their morphology was observed via electron microscopy, and their sensitivity to ultraviolet radiation and ethanol, tolerance to pH and temperature, optimal multiplicity of infection n (MOI) and one-step growth curve were determined. The lysis range was assessed through host spectrum assays. The genomic features, functional annotation and protein structure prediction were analyzed on bases of whole genome sequencing, the phylogenetic tree and average nucleotide identity heatmaps were constructed, and the comparative genomic analysis was performed. RESULTS There was 1 strain of multidrug-resistant P. aeruginosa isolated and its specific phage with about 60 nm of head and 301 nm of tail; 30 min of ultraviolet radiation or acting with ethanol at concentration more than 50% could inactivate the strain; it remained stable in environment with pH value ranging between 2.0 and 12.0 or the temperature ranging between 4 and 50 ℃; the optimal MOI was 0.00001, with a latent period 70 min, the amount of lysis approximately 50 plaque forming unit (PFU)/cell. YN02 specifically lysed P. aeruginosa only. The genome was 93 989 bp in length with a GC content of 0.55. A total of 128 open reading frames (ORFs) were predicted, and YN02 was identified as a new member of the Samunavirus genus. No antimicrobial resistance genes, virulence factors or lysogeny-related proteins were detected. CONCLUSION The isolated phage YN02 exhibits high intolerance to ethanol, resistance to high temperature and pH and strict host specificity, which may provide scientific bases for phagotherapy, prevention and treatment of multidrug-resistant P. aeruginosa.