Klebsiella pneumoniae is a significant threat to global health. At present, the methods of K. pneumoniae detection are culture-based and instrument-dependent and are not suitable for rapid diagnostic. Klebsiella pneumoniae (K. pneumoniae) is one of the most common pathogens causing nosocomial infection. A rapid, accurate, and convenient detection method is required for early diagnosis and directed therapy of K. pneumoniae infection. CRISPR-top (CRISPR-mediated testing in one pot) is a LAMP-CRISPR-based nucleic acid detection platform, which integrates target preamplification with CRISPR/Cas12b-based detection into a one-pot reaction mixture, performed at a constant temperature. In this study, we established the K. pneumoniae CRISPR-top assay to precisely identify K. pneumoniae at 56 degrees C within 60 min. The reaction mixture with 0.53 mu M (each) FIP and BIP, 0.27 mu M LF, 0.13 mu M (each) F3 and B3, and 2 mu M ssDNA fluorescence probe was determined as the optimal reaction system of our assay. The limit of detection of this assay is 1 pg genomic DNA (equivalent to 160 K. pneumoniae cells and 1.6 x 10(5) CFU/mL for samples) per reaction, which is 10-fold more sensitive than LAMP. Up to 105 strains composed of K. pneumoniae clinical isolates and non-K. pneumoniae strains were correctly identified by our assay. A total of 58 sputum samples collected from patients with respiratory symptoms were used to evaluate the diagnostic performance of the K. pneumoniae CRISPR-top assay. As a result, the K. pneumoniae CRISPR-top assay yielded 100% (33/33) specificity and 96% (24/25) sensitivity, as well as a positive predictive value of 100% (24/24) and a negative predictive value of 97.1% (33/34), which were all higher than LAMP detection. In conclusion, the K. pneumoniae CRISPR-top assay developed in this study is a simple, rapid and ultra-specific method to detect K. pneumoniae. IMPORTANCE Klebsiella pneumoniae is a significant threat to global health. At present, the methods of K. pneumoniae detection are culture-based and instrument-dependent and are not suitable for rapid diagnostic. This study reports K. pneumoniae CRISPR-top assay, which can precisely identify K. pneumoniae using nucleic acids of pure cultures or clinical samples in one pot with one fluid-handling step. The K. pneumoniae CRISPR-top reaction can be completed within 60 min at a constant temperature, thus specific instruments are not required. Our results show that CRISPR-top assay yields enormous advantages compared with LAMP detection. The K. pneumoniae CRISPR-top assay can be a high-efficiency alternative tool for rapid and accurate diagnosis of K. pneumoniae infection, especially in resource-limited settings.