Mapping the spatial locations of gut bacteria in their native environment enhances our understanding of bacteria–host interactions and the physiological and pathological roles these microbes play. However, the intricate composition of bacterial communities in millimeter-scale intestinal tissues presents a great challenge for in situ imaging of their spatial distributions. To address this, we introduce a three-dimensional (3D) imaging strategy that combines a fluorescent tetrapeptide (TetraAA-AcLys) metabolic labeling probe with a tissue clearing protocol. This method enables high-resolution visualization of the microbiota within intact colon, allowing for clear observation of the 3D distribution of gut bacteria across various sections, without interference from host tissues. Moreover, 3D quantitative analysis of the labeled bacteria in a enteritis model reveals their penetration into the mucus layer in colon, highlighting the technique’s potential for studying gut microbiota biogeography in health and disease. This 3D imaging method offers valuable spatial insights into the dynamic relationship between the microbial community and its host.