Background 4‐Hydroxyphenylpyruvate dioxygenase (HPPD), playing a critical role in vitamin E and plastoquinone biosynthesis in plants, has been recognized as one of the most important targets for herbicide discovery for over 30 years. Structure‐based rational design of HPPD inhibitors has received more and more research interest. However, a critical challenge in the discovery of new HPPD inhibitors is the common inconsistency between molecular‐level HPPD‐based bioevaluation and the weed control efficiency in fields, due to the unpredictable biological processes of absorption, distribution, metabolism, and excretion. Results In this study, we developed a fluorescent‐sensing platform of efficient in vivo screening for HPPD‐targeted herbicide discovery. The refined sensor has good capability of in situ real‐time fluorescence imaging of HPPD in living cells and zebrafish. More importantly, it enabled the direct visible monitoring of HPPD inhibition in plants in a real‐time manner. Conclusion We developed a highly efficient in vivo fluorescent screening method for HPPD‐targeted herbicide discovery. This discovery not only offers a promising tool to advance HPPD‐targeted herbicide discovery, but it also demonstrates a general path to develop the highly efficient, target‐based, in vivo screening for pesticide discovery. © 2022 Society of Chemical Industry.