The　gut　bacteria　not　only　play　a　crucial　role　in　maintaining　human　health　but　also　exhibit　close　associations　with　the　occurrence　of　numerous　diseases.　Understanding　the　physiological　and　pathological　functions　of　gut　bacteria　and　enabling　early　diagnosis　of　gut　diseases　heavily　relies　on　accurate　knowledge　about　their　in　vivo　distribution.　Consequently,　there　is　a　significant　demand　for　noninvasive　imaging　techniques　capable　of　providing　real-time　localization　information　regarding　gut　bacteria.　In　this　work,　we　developed　a　second　near-infrared　(NIR-II)　fluorescent　nanoprobe　labeling-based　visualization　strategy　for　real-time　tracking　of　the　biodistribution　of　Gram-negative　bacteria　in　the　gastrointestinal　tract　of　mice.　By　utilizing　positively　charged　silver　sulfide　quantum　dots　(Ag2S　QDs)　as　NIR-II　nanoprobes　and　exploiting　electrostatic　interactions　to　efficiently　label　the　Gram-negative　probiotic　Escherichia　coli　Nissle　1917　with　negative　surface　charges,　we　have　achieved　rapid　and　effective　labeling.　Leveraging　the　exceptional　NIR-II　fluorescent　performance　of　Ag2S　QDs,　our　approach　enables　high　spatiotemporal　resolution　visualization　via　NIR-II　imaging　in　mouse　gastrointestinal　areas　where　Ag2S　QD-labeled　probiotics　are　present,　facilitating　real-time　in　vivo　tracking　capabilities　for　these　labeled　probiotics.　This　work　not　only　establishes　a　powerful　intestinal　bacterial　imaging　strategy　but　also　introduces　novel　concepts　for　constructing　nanomaterial-bacteria　hybrid　systems.　©　2025　The　Authors.　Published　by　American　Chemical　Society.