AbstractIn this study, fenugreek seed proteins were extracted using ultrasonic‐assisted extraction with varying solid:solvent ratios (20–60 g/L) and sonication amplitudes (30%–80%) to determine optimal conditions for the highest extraction yield. The functional, structural, and nutritional characteristics of the protein isolates of fenugreek seeds were investigated. The highest yield (98.74 ± 0.49%) was achieved at a solid:solvent ratio of 43.83 g/L and an amplitude of 67.51%. The coagulated protein values of fenugreek seed protein isolates ranged from ~15.8% to 31.2%, water‐holding capacities ranged from ~2.2 to 3.2 g/g, oil‐holding capacities ranged from ~2.6 to 4.1 g/g, foaming capacities ranged from ~16.3% to 21.3%, foam stabilities ranged from ~59.7% to 78.1%, emulsion stabilities ranged from ~30.2 to 34.5 min, emulsion activities ranged from ~73.8 to 76.8 m2/g, and emulsion capacities ranged from ~26.9% to 30.5% under different extraction conditions. SDS‐PAGE analysis revealed three distinct bands (46, 59, and 80 kDa) for the protein isolates. FT‐IR spectroscopy showed a high presence of β‐sheet structures. The amino acid composition analysis of fenugreek seed protein isolates was determined, revealing richness in essential amino acids (317.97 g amino acid/kg protein isolate). In addition, cupcakes enriched with protein isolates (5%, 10%, and 20% as flour substitutes) were produced, and quality properties such as color change, browning index, moisture content, water activity, baking yield, bulk density, hardness, volume, symmetry, and uniformity indexes were determined. The application of protein isolates in cupcake production demonstrated the potential of fenugreek seeds as valuable ingredients for enhancing the nutritional profile of bakery products.