<jats:title>Abstract</jats:title>
          <jats:p>This study focuses on optimizing a single-step ultrasonic-assisted aqueous enzymatic extraction (UAE-AEE) to valorize the solid residue from Horchata de Chufa (SRH). Initial characterization of dried SRH revealed 20.6 ± 0.5% fat and 70.4 ± 0.6% carbohydrates. A Box-Behnken Design was used to develop an eco-friendly method for extracting the oil content using distilled water and cellulase, hemicellulase, and pectinase enzymes. The optimal conditions for the extraction of the oil fraction were 30 min, 8 cycles, and 0.5 g of SRH, yielding 14.56 ± 0.02%. The oily fraction (OF) and fibrous sediment phase (FF) were thoroughly characterized. UAE-AEE enhanced mass transfer, releasing oil and starch granules, as confirmed by scanning electron microscopy. Major fatty acids in the OF included oleic (60.4 ± 1.3%), palmitic (11.4 ± 0.9%), linoleic (6.5 ± 0.4%), and stearic (2.31 ± 0.15%), with a saponification value of 213 ± 7 mg KOH g<jats:sup>−1</jats:sup>. The main monosaccharides were glucose, manose, galactose, and fructose, with interesting content in potassium, magnesium, and calcium. The total phenolic content of the OF (213 ± 7 mg GAE 100 g<jats:sup>−1</jats:sup>) was higher than that of oil extracted by other methods, with notable in vitro antioxidant activity measured by CUPRAC, ABTS, FRAP, and DPPH assays. The OF demonstrated potential as a stabilizer in horchata-like beverages at 2% wt., while the FF, rich in α-cellulose and lignin, could be a valuable source of dietary fiber and prebiotics. FTIR and thermogravimetric analyses confirmed the chemical composition of the fractions. This study provides an eco-friendly method to valorize horchata by-products as rich source of oil and fiber and supports the shift toward sustainable and efficient extraction methods for food applications.
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