Gut microbiota, widely populating the mammalian gastrointestinal tract, plays an important role in regulating diverse pathophysiological processes by producing bioactive molecules. Extensive detection of these molecules contributes to probing microbiota function but is limited by insufficient identification of existing analytical methods. In this study, a systematic strategy was proposed to detect and annotate gut microbiota-related metabolites on a large scale. A pentafluorophenyl (PFP) column-based liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was first developed for high-coverage analysis of gut microbiota-related metabolites, which was verified to be stable and robust with a wide linearity range, high sensitivity, satisfactory recovery, and repeatability. Then, an informative database integrating 968 knowledge-based microbiota-related metabolites and 282 sample-sourced ones defined by germ-free (GF)/antibiotic-treated (ABX) models was constructed and subsequently used for targeted extraction and annotation in biological samples. Using pooled feces, plasma, and urine of mice for demonstration application, 672 microbiota-related metabolites were annotated, including 21% neglected by routine nontargeted peak detection. This strategy serves as a useful tool for the comprehensive capture of the intestinal flora metabolome, contributing to our deeper understanding of microbe-host interactions.