A novel high-throughput aptamer microarray fluorescent method based on thioflavin T (ThT) was established for the sensitive detection of phoxim, parathion, fensulfothion, and isocarbophos. In this work, the aptamers in binding buffer tended to have the antiparallel G-quadruplex structure, which can bind ThT and release its potential fluorescence signal. However, when the organophosphorus pesticides (OPs) were present, partial aptamers preferred to bind them, forcing the displacement of ThT from the G-quadruplex and resulting in the significant decrease in fluorescence signal. Under optimal experimental conditions (12T spacer, 300 nM aptamer, and 80 μM ThT), the OP aptamer microarray has low limits of detection of 25.4 ng/mL for phoxim, 12.0 ng/mL for parathion, 7.7 ng/mL for fensulfothion, and 9.9 ng/mL for isocarbophos. The accuracy and reliability of the method is further verified by testing the recovery rate of OPs spiked in two different complicated sample matrices (pears and radishes). It is worth mentioning that not only the developed aptamer microarray technology has low sensitivity and a broad spectrum, but it also allows for high-throughput and rapid analysis of a variety OPs, which overcomes some of the shortcomings of other OP detection methods.