Trehalose is an autophagy-promoting disaccharide, which can improve and delay chronic diseases like neurodegenerative diseases and atherosclerosis, but its bioavailability is severely restricted by endogenous trehalase in mammals. Trehalase inhibitor is a promising and effective way to enhance trehalose bioavailability by preventing trehalose from hydrolyzing. However, previously reported trehalase inhibitors still face safety of long-term use and promiscuous inhibition on intestinal glycosidases. This study carried out a high-throughput virtual screening through molecular pool-based molecular docking combined with in vitro inhibition experiments to screen trehalase inhibitors naturally derived from foods. Out of 1 769 small molecules, which include 115 analogs of trehalose, natural monosaccharides, disaccharides, trisaccharides, imidazoles and their derivatives, as well as 20 natural amino acids and their 400 dipeptides, isomaltose, α-isomaltulose, and isomaltitol exhibited the best inhibitory activities, beyond as traditional sweetener and prebiotic. Best of all, isomaltose showed the half maximal inhibitory concentration (IC50) and inhibition constant (Ki) values on trehalase of 5.59 and (2.176 0 ± 0.343 1) mmol/L, respectively. Moreover, isomaltose was resistant to the simulated digestive environment and did not affect intestinal glycosidases such as α-glucosidase and glucoamylase, making it a reliable edible candidate for a trehalase inhibitor. This study provides new insights into the virtual screening-based identification of new food-derived trehalase inhibitors for enhanced integrity and bioavailability of orally administered trehalose, especially repurposing a prebiotic for another new use as trehalase inhibitor.