Peanut (Arachis hypogaea L.), an important oilseed crop, is primarily cultivated in arid and semi-arid regions, where water stress is a major constraint on yields. However, the mechanisms underlying stomatal response to drought stress in peanut remain largely unknown. In this study, a field experiment with controlled water regimes was carried out to investigate the stomatal traits of peanuts. Compared with the 70 % field capacity (FC) control, both conditions of 55 % FC and 45 % FC induced stomatal closure. However, only the 45 % FC treatment significantly enhanced the instantaneous water use efficiency (WUEi) of the plants. Transcriptome and metabolome analysis revealed a significant enrichment of flavonol biosynthesis in guard cells under 45 % FC. Further research showed that exogenous application of quercetin or kaempferol significantly reduced the reactive oxygen species (ROS) accumulation in guard cells and promoted stomatal closure under drought stress. Furthermore, exogenous flavonols spraying was found to stimulate the abscisic acid (ABA) biosynthesis by upregulating the transcription of 9-cis-epoxycarotenoid dioxygenase gene (AhNCED1). Together, these results suggested that the flavonol pathway was activated in guard cells of peanut under drought stress condition, which facilitate the ABA biosynthesis to induce stomatal closure, and exogenous flavonols spraying further enhanced this effect.