Transketolase (TK) was identified as a new target for the development of novel herbicides. In this study, a series of naphthalimide−aroyl hybrids were designed and prepared based on TK as a new target and tested for their herbicidal activities. In vitro bioassay showed that compounds 4c and 4w exhibited stronger inhibitory effects against Digitaria sanguinalis (DS) and Amaranthus retroflexus (AR) with the inhibition over 90% at 200 mg/L and around 80% at 100 mg/L. Also, compounds 4c and 4w exhibited excellent postemergence herbicidal activity against DS and AR with the inhibition around 90% at 90 g [active ingredient (ai)]/ha and 80% at 50 g (ai)/ha in the greenhouse, which was comparable with the activity of mesotrione. The fluorescent quenching experiments of At TK revealed the occurrence of electron transfer from compound 4w to At TK and the formation of a strong exciplex between them. Molecular docking analyses further showed that compounds 4w exhibited profound affinity with At TK through the interaction with the amino acids in the active site, which results in its strong inhibitory activities against TK. These findings demonstrated that compound 4w is potentially a lead candidate for novel herbicides targeting TK.