Due to the large-scale use of organophosphorus pesticides (OPs), their contamination in the environment is widespread throughout the world, especially in developing countries. With increasing public concern, there is an urgent requirement for simple, rapid, high-throughput and highly sensitive analytical methods for the screening of OP residues in the environment. In this work, a monoclonal antibody-based sensitive one-step direct competitive time-resolved fluorescence immunoassay (TRFIA) with broad specificity to a class of OPs was developed. After optimization of the assay conditions, this method can detect thirteen OPs with a limit of detection below 10 ng mL⁻¹. The recoveries of OPs from spiked environmental water samples ranged from 74.8% to 121.3%, with the coefficient of variation (CV) ranging from 6.4% to 15.1%. The correlation coefficient between the TRFIA results and standard HPLC-MS/MS results was 0.964. The proposed TRFIA was capable of high-throughput analysis of a large number of samples prior to chromatographic analysis, with good sensitivity, simplicity and rapidity.