It was necessary to develop simple and reproducible diagnostic tools for endangered species, and their associated pathogens. Klebsiella pneumoniae was the main pathogenic bacterium leading to intestinal disease within Giant Panda (Ailuropoda melanoleuca). A noninvasive method for the specific detection of K. pneumoniae in Giant Pandas' s feces was developed and evaluated in this study. The 368-bp phoE gene was selected for specificity, and 2 types of K. pneumoniae strains in conjunction with 16 non-K. pneumoniae strains were evaluated. The Key step of noninvasive sampling procedure was to remove the inhibitors of Polymerase Chain Reaction (PCR), which was based on that silica powders could bind pathogen DNA at the conditions of high concentration of kalium iodide and neutral pH. Before PCR cycle, the bound DNA was washed with 80% ethanol and eluted with a diluted buffer. To validate the PCR assay, an experiment was performed with both artificially contaminated and natural fecal matter. The results indicated that the method could detect specifically K. pneumoniae from Giant Pandas's feces with considerable sensitivity.