Quantum dots (QDs), luminescent semiconductor nanocrystals, have the potential to simplify the performance of multi-analyte analysis. In this work, a novel indirect competitive fluorescence-linked immunosorbent assay (FLISA) based on QDs for the detection of clothianidin and thiacloprid simultaneously in environmental and agricultural samples were performed in a single well of the microtiter plate. The QDs-labeled antibody conjugates, which consists of CdSe/ZnS core–shell QDs and polyclonal antibodies, were prepared through the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) coupling method. Under optimal conditions, the half-maximal inhibition concentration (IC₅₀) and the limit of detection (LOD, IC₁₀) of the FLISA were 12.5 and 0.3 ng mL⁻¹ for clothianidin, and 9.27 and 0.4 ng mL⁻¹ for thiacloprid, respectively. The cross-reactivities of the FLISA with the analogues of clothianidin and thiacloprid were negligible except for dinotefuran (10.4% for clothianidin). The spiked recoveries were 73.3% to 113.1% with relative standard deviations (RSDs) of 2.7 to 13.3% for the detection of clothianidin and thiacloprid in river water, soil, cabbage, rice and tomato. Furthermore, the results of FLISA for the authentic samples correlated well with those obtained by HPLC. The proposed FLISA is a satisfactory tool for rapid, sensitive, and quantitative detection of two pesticides simultaneously in agricultural and environmental samples. This study provides a strategy for the development of QDs-based fluoroimmunoassay for the quantitative analysis of two pesticides.