Ulva green macroalgae or sea lettuce are rich sources of protein with nutritional benefits that promote health as a future plant-based functional ingredient in the food industry. Alkaline pretreatment improved ultrasonic-assisted protein extraction from Ulva rigida biomass. Parameters affecting ultrasonic-assisted extraction of protein were type of solvent, biomass-solvent ratio, biomass preparation and extraction cycle. In vitro digestibility was evaluated from oven- and freeze-dried biomass. Results showed highest concentration and extraction yield of protein from U. rigida using alkaline rather than acid and distilled water. A high biomass–solvent ratio at 1:10 or 0.1 g mL⁻¹ increased protein extraction. Higher alkaline concentration increased protein extraction. Highest protein extractability was 8.5% dry matter from freeze-dried U. rigida biomass, with highest protein extraction and antioxidant activity from extraction of U. rigida macroalgae at high alkaline concentrations. U. rigida macroalgae oven-dried biomass presented suitable human digestibility. Efficient pretreatment of U. rigida maximized protein hydrolysate and bioactive peptide production for wide-ranging applications.