The excessive use of pesticides is an urgent issue facing environmental sustainability and human health. In this study, a uniform dispersion size, good fluorescence performance and mesoporous structure of a ratiometric fluorescent probe were constructed for nicosulfuron detection. A solvent-free in situ solid-phase synthesis method was used to encapsulate biomass carbon dots within mesoporous silica (CDs@mSiO₂), followed by the modification of l-cysteine-modified manganese-doped zinc sulfide quantum dots (ZnS:Mn QDs), to construct a ratiometric fluorescent probe for highly sensitive and selective detection of nicosulfuron. This design effectively prevents the aggregation of CDs and reduces interference between the two fluorescent signals. Nicosulfuron detection had a low detection limit of 0.082 μM. Density functional theory calculations were carried out to uncover the specific interactions between nicosulfuron and ZnS:Mn QDs. The process of fluorescence quenching is ascribed to photoinduced electron transfer. This work offers a special strategy to produce a ratiometric fluorescent probe and illustrate the mechanism, which is crucial for sensing and environmental engineering.