The exploration of new protein sources will have a significant impact on future food development. This study examined the effects of various factors involved in alkaline ultrasonic extraction on Euglena protein yield by employing the single factor, Plackett-Burman, and response surface methods. UV-Vis spectroscopy, FT-IR spectroscopy, and automated amino acid analysis were adopted to evaluate the functional group composition, secondary structure, and amino acid profile of Euglena protein. Additionally, the functional properties, such as water absorption, oil-holding capacity, and emulsification capacity, were studied. The optimal extraction parameters are as follows: a solid-to-liquid ratio of 1:20, an ultrasonic power of 100 W, an alcohol-to-base ratio of 1:3.5, a sodium hydroxide concentration of 0.35 mol/L, an extraction temperature at 40 ℃, an extraction time of 120 min, and an ultrasonic treatment time of 5 min. These conditions result in an extraction yield of 93.81%. Structural characterization reveals that the secondary structure of Euglena protein primarily consists of β-turns and β-sheets, accounting for 98.80% of the total structure. Essential amino acids account for 66.09% of the total composition, with hydrophobic amino acids constituting 67.81%. At 60 ℃, Euglena protein exhibits the highest maximum water absorption, reaching up to 13.19 g/g. Meanwhile, the highest oil-holding capacity of 7.33 g/g is observed at 40 ℃. Euglena protein demonstrates good emulsification capacity, emulsification stability, foaming capacity, and foam stability, with percentages of 34%, 64.62%, 53.90%, and 81.79%, respectively. This study utilized alkaline ultrasonic extraction to enhance the extraction yield of Euglena protein, laying the groundwork for its application as a novel protein source.