Real-time localization and microbial activity information of indigenous gut microbiota over an extended period of time remains a challenge with existing visualizing methods. Herein, we report a metabolic fluorine labeling (MEFLA)-based strategy for monitoring the dynamic gut microbiota via ¹⁹F magnetic resonance imaging (¹⁹F MRI). In situ labeling of different microbiota subgroups is achieved by using a panel of peptidoglycan-targeting MEFLA probes containing ¹⁹F atoms of different chemical shifts, and subsequent real-time in vivo imaging is accomplished by multiplexed hotspot ¹⁹F MRI with high sensitivity and unlimited penetration. Using this method, we realize extended visualization (> 24 h) of native gut microbes located at different intestinal sections and semi-quantitative analysis of their metabolic dynamics modulated by various conditions, such as the host death and different β-lactam antibiotics. Our strategy holds great potential for non-invasive and real-time assessing of the metabolic activities and locations of the highly dynamic gut microbiota.