Hyperoside is a natural flavonol glycoside that widely exists in many tissues (especially the aerial parts) of diverse plants. Remarkably, hyperoside has exhibited prominent biological activities against a variety of diseases including cancer, myocardial ischemia, stroke, depression, Alzheimer’s disease, Perkins syndrome, etc., and has very low toxicity in the effective dose range. In view of its notable bioactivities and pharmacological effects, hyperoside is considered to possess a promising application prospect in food and medicinal industry. Based on latest hyperoside’s research progress retrieved from various databases, including Web of Science, Google Scholar, PubMed, Scopus, etc., this review aims to depict current research status of hyperoside in the aspect of resources, synthesis pathways, physicochemical properties, metabolism, pharmacological activities, clinical trials and human studies, toxicology and safety, marketed products, and patents. Moreover, this review also summarizes and discusses the opportunities and challenges associated with hyperoside, which could help us more thoroughly and comprehensively understand hyperoside as a potential candidate for drug discovery, and provide a theoretical foundation for the further investigation of hyperoside.