〔Abstract〕 Objective To construct the K64 capsule depolymerase recombinant protein, Dep44, and investigate its potential application against carbapenem-resistant Klebsiella pneumoniae(CRKP) infections. Methods The depolymerase-encoding phage vB＿Kpn＿HF1013(GenBank: PP803128) was isolated and genomically analyzed to screen for candidate depolymerases. The recombinant protein Dep44 was constructed and functionally verified for depolymerase activity. Dep44 sensitive range was validated and Dep44 antimicrobial activity was assessed by biofilm disruption and serum sterilization assays. Results The tail spike protein of phage vB＿Kpn＿HF1013 exhibited depolymerase activity and recombinant protein Dep44 specifically degraded K64 CRKP capsule. Biofilm eradication assays demonstrated that recombinant Dep44 at both 2 μg/mL and 10 μg/mL significantly disrupted bacterial biofilms relative to the control. Serum bactericidal assays showed that Dep44 exhibited synergistic activity with serum, dependent on the complement system, as Dep44 alone lacked bactericidal properties. Conclusion Dep44 effectively targets and degrades K64 CRKP capsule, disrupts biofilms, and enhances serum bactericidal activity, highlighting its potential for managing K64 CRKP infections and clearing biofilms from medical devices.