Abstract: Lotus (Nelumbo) is an economically important aquatic crop and contains abundant bioactive compounds, such as flavonoids, alkaloids, and polysaccharides. More than 100 flavonoids have been identified in lotus, including rutin, quercetin, and chrysin, which exhibit various antibacterial, anti-obesity, and antioxidant medicinal properties. In this review, we introduce the types of flavonoids in lotus and various effective extraction and identification methods, including microporous resin chromatography (MPRC) and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Moreover, the functional activities of flavonoids in lotus and their latest applications, including disease treatment and functional product development, are further summarized. This review provides a reference for future research and the comprehensive utilization of lotus flavonoids.