A monoclonal antibody (mAb) was raised against tebuconazole (TEB) using a hapten where the -chloro substituent of the TEB molecule was replaced with a long-chain carboxylic acid. The resulting mAb showed high sensitivity and specificity against TEB characterized by ELISA with a half-maximal inhibitory concentration (IC) of 0.19 ng mL and with cross-reactivity (CR) values below 0.01% to several analogues of triazole fungicides. On the basis of the mAb produced, a quantum dot beads-based fluorescence immunochromatographic test strip assay (QBs-FITSA) was developed for rapid and sensitive detection of TEB in agricultural product samples. The QBs-FITSA exhibited a linear detection range from 0.02 to 1.25 ng mL with a limit of detection (LOD) of 0.02 ng mL. Furthermore, using produced mAb, multiple high-throughput rapid immunoassay formats could be achieved as a convenient monitoring tool for evaluation of human and environmental exposure to TEB.