Cost-effective and user-friendly quantitation at points-of-need plays an important role in food safety inspection, environmental monitoring, and biomedical analysis. This study reports a stand-alone smartphone-based fluorospectrophotometer (the SBS) installed with a custom-designed application (the SBS-App) for on-site quantitation of pesticide using a ratiometric sensing scheme. The SBS can collect fluorescence emission spectra in the wavelength range of 380-760 nm within 5 s. A ratiometric fluorescence probe is facilely prepared by directly mixing the blue-emissive carbon nanodots (the Fe3+-specific fluorometric indicator) and red-emissive quantum dots (the internal standard) at a ratio of 11.6 (w/w). Based on the acetylcholinesterase/choline oxidase dual enzyme-mediated cascade catalytic reactions of Fe2+/Fe3+ transformation, a ratiometric fluorescence sensing scheme is developed. The practicability of the SBS is validated by on-site quantitation of chlorpyrifos in apple and cabbage with a comparable accuracy to the GC-MS method, offering a scalable solution to establish a cost-effective surveillance system for pesticide pollution. Display omitted •The SBS is developed for the on-site collection of fluorescence spectra•The SBS-App is programmed for spectrum reconstruction and quantitative analysis•A ratiometric fluorescence sensing scheme is designed for chlorpyrifos quantitation Environmental science; Applied sciences