© 2020 Elsevier Ltd In this work, in-syringe membrane solid-phase extraction (MSPE) device was fabricated for the on-site sampling of sulfamethoxazole (SMX) in food samples followed by solid-phase fluorescence spectra analysis. The samples and fluorescamine (FA) were added to a syringe for derivation. Then, the derivative of SMX was extracted by a membrane in the syringe SPE device. Subsequently, the derivative on the membrane was measured immediately without additional elution procedure. The method was successfully applied in plasma, milk, and egg samples for the trace SMX detection, with the recovery of 98%–102%, RSDs from 1% to 6%. Compared with liquid chromatography, direct detection of the concentrated analyte significantly improved the sensitivity. Moreover, fluorescamine made it unnecessary to separate SMX from the interference. Consequently, it was a time-saving, low-cost, and easy-operation method, which demonstrated the potential of in-syringe SPE as a promising candidate for on-site analysis.