Recent advancements in analytical strategies have enabled the efficient extraction and characterization of bioactive compounds from agri-food bio-residues, emphasizing green chemistry and circular economy principles. This review highlights the valorization of several agri-food bio-residues for the extraction of high-value-added bioactive compounds, particularly polyphenols, tocopherols, carotenoids, and fatty acids, as a biorefinery approach. To this end, the adoption of environmentally friendly extraction technologies is essential to improve performance, reduce energy consumption, and minimize costs. This study therefore examines emerging methodologies such as supercritical fluid extraction, pressurized liquid extraction, pulsed electric fields, and matrix solid-phase dispersion, highlighting their advantages and limitations. Additionally, the chemical characterization of these bioactive compounds is explored through spectrophotometric and high-resolution chromatographic techniques, crucial for their accurate identification and quantification. This is complemented by an analysis of bioactivity assays evaluating antioxidant, antimicrobial, anticancer, neuroprotective, and anti-inflammatory properties, with a focus on their applications in the food, pharmaceutical, and cosmetic industries. However, the analytical control of toxic compounds, such as alkaloids, in these bio-residues is undoubtedly needed. Ultimately, this approach not only promotes sustainability but also contributes to the development of eco-friendly solutions in various industries.