Abstract: Objective: To establish a loop-mediated isothermal amplification (LAMP) method for the detection of Klebsiella pneumoniae (Kpn) in sputum of patients with respiratory infection. Methods: Four specific primers were designed based on phoE gene of Kpn, and the concentrations of Mg²⁺ and betaine in reaction conditions were optimized. After the LAMP assay for the detection of Kpn was established, its specificity and sensitivity were evaluated. A total of 308 sputum samples from the patients with respiratory infection were collected and detected by the bacterial culture method, LAMP assay and DNA sequencing technology, respectively, to evaluate the detection performance of the LAMP assay. Results: The optimal concentrations of Mg²⁺ and betaine were 8 mmol/L and 0.4 mmol/L, respectively. The established LAMP assay was highly specific and had no cross reaction with other strains, and its sensitivity was 10⁴ CFU/mL. The detection rate of Kpn (53.89%) by the LAMP assay was significantly higher than that by the bacterial culture method (42.20%, χ²=161.65, P＜0.05). The coincidence rates between the LAMP assay and the bacterial culture method were 96.15%, 100% and 65.25%, respectively, for Kpn positive samples, without bacterial infection samples and non-Kpn bacteria positive samples. Forty-six samples with inconsistent results were verified by DNA sequencing, and the coincidence rate of the LAMP assay and DNA sequencing was 86.95% (40/46). 131 samples were detected by the LAMP assay and DNA sequencing, respectively, and they had excellent consistency by Kappa test (Kappa coefficient=0.817). Conclusion: Compared with the bacterial culture method, the established LAMP assay has the advantages of high accuracy, high sensitivity, strong specificity, and time-saving reaction, which may be used as a rapid detection method for Klebsiella pneumoniae.