Direct fluorescence detection of amoxicillin (AMX) is highly desired for food and environment safety but remains a formidable challenge because of its low conjugation degree and stabilization effects on excitons. In this work, we proposed a direct fluorometry for AMX through modulating its fluorescence properties by utilizing sulfur nanodots (S-dots) as nanoreactors. S-dots provided both confined carrier and reactants (polyethylene glycol (PEG) and sulfite ions) to modulate the fluorescence properties of AMX, achieving a 277-fold increase on the fluorescence intensities. PEG and SO32- interacted with AMX through both covalent and hydrogen bounds, which stabilized the excited states, decreased the chances of nonradiative path and activated the radiative decay. The ultrasmall sized S-dots also provided confinement carries for promoting the reactivity between AMX and surface function groups. Quantitative detection of AMX in commercial milk samples was realized through direct recording the fluorescence signals, achieving a limit of detection of 159 nM. The reported results provided a new idea to design fluorescence assays for extremely weak emissive molecules, showing great potential in food safety analysis, environmental and healthy monitoring.