Cruciferous sprout is a new form of vegetable product that is gaining attention due to its high content of bioactive compounds such as glucosinolates. As a diverse class of phytochemicals, glucosinolates and glucosinolate-derived products such as isothiocyanates are popular targets for scientific research because of their health associated benefits. The present review highlights the unique properties of beneficial glucosinolates and bioactive derivatives, the biosynthetic and metabolic regulatory networks of glucosinolates, and their role in cruciferous sprouts for producing health-promoting glucosinolates biofortification benefits along the entire agro-food chain. Glucosinolates and their bioactive derivatives have been extensively studied for their beneficial effects on human health. Among various plant products, cruciferous sprouts are more suitable as a functional food due to their high content of health-promoting glucosinolates and bioactive derivatives. This review describes the unique properties of glucosinolates and their bioactive derivatives contained in cruciferous sprouts with potential health benefits. We discussed the glucosinolate metabolic pathway as well as their regulatory mechanisms. Furthermore, we proposed to apply the integrative and sustainable treatment approaches in cruciferous sprout industry including breeding for glucosinolates-biofortified cultivars, pre-harvest treatment to improve glucosinolates accumulation, followed by postharvest handlings and processing methods for benefit glucosinolates retention along the whole agro-food chain to fight health-promoting glucosinolates deficiency in human daily diet. This review presents a comprehensive overview of effective health-promoting glucosinolates biofortification in cruciferous sprouts along the entire agro-food chain, which is potential in the sustainable cruciferous sprouts industry as well as significance in human nutrition.