Flavonoids are a group of plant constituents called phenolic compounds and correspond to the nonenergy part of the human diet. Flavonoids are found in vegetables, seeds, fruits, and beverages such as wine and beer. Over 7000 flavonoids have been identified and they have been considered substances with a beneficial action on human health, particularly of multiple positive effects because of their antioxidant and free radical scavenging action. Although several studies indicate that some flavonoids have provident actions, they occur only at high doses, confirming in most investigations the existence of anti-inflammatory effects, antiviral or anti-allergic, and their protective role against cardiovascular disease, cancer, and various pathologies. Flavonoids are generally removed by chemical methods using solvents and traditional processes, which besides being expensive, involve long periods of time and affect the bioactivity of such compounds. Recently, efforts to develop biotechnological strategies to reduce or eliminate the use of toxic solvents have been reported, reducing processing time and maintaining the bioactivity of the compounds. In this paper, we review, analyze, and discuss methodologies for biotechnological recovery/extraction of flavonoids from agro-industrial residues, describing the advances and challenges in the topic.