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A novel high-performance liquid chromatography-fluorescence analysis in combination with in situ degradation-derivatization (ISD-D) technique was developed for simultaneous determination of seven organophosphorus thioester pesticides (OPTPs) in tea. The ISD-D technique was based on degradation of OPTPs by a nucleophilic substitution reaction between phenylbutane-1,2,3-trione-2-oxime and OPTPs, which can give thiol degradation products (DPs). The thiol DPs obtained were derivatized with the novel derivatization reagent N-(4-(carbazole-9-yl)-phenyl)-N-maleimide (NCPM) in a syringe. Attractively, NCPM itself did not fluoresce, whereas the derivatives of the thiol DPs fluoresced intensely, with excitation and emission maxima at 290 nm and 368 nm, respectively, which extraordinary reduced the background interference and increased the detection sensitivity for thiol DPs. Excellent linearity (R2 > 0.995) for all OPTPs was achieved, with limits of detection and limits of quantitation ranging from 0.23 to 0.45 μg/kg and from 0.75 to 1.43 μg/kg, respectively. Satisfactory recoveries ranging from 90.5% to 96.0% were obtained for all OPTPs. The ISD-D technique provided a novel and sensitive strategy for quantitation of trace amounts of OPTPs in real samples. Graphical abstract ᅟ.