Microalgae are gaining attention as promising natural sources of bioactives due to their rich nutrient content. This study investigates the effects of ultrasound-assisted extraction (UAE) parameters (i.e., solvent composition, temperature, solid-to-solvent ratio, and time) on the extraction of phenolic compounds from Tetraselmis tetrahele. The UAE parameters were determined at a solid-to-solvent ratio of 1:75, a temperature of 25 °C, and an extraction time of 15 min with 75% ethanol as the solvent. Phenolic profile, total phenolic content, total flavonoid content, radical scavenging activity of DPPH and ABTS, photosynthesis pigment (chlorophyll a, b, and total carotenoid), anti-collagenase activity, and metabolic profile of Tetraselmis tetrahele extract obtained using UAE were determined. Tetraselmis tetrahele extracts obtained using the selected UAE method demonstrated significantly higher (p < 0.05) levels in gallic acid (1.81%), 4-hydroxybenzoic acid (7.0%), total phenolic content (84.0%), total flavonoid content (56.2%), ABTS (101.3%), chlorophyll a (119.4%), chlorophyll b (174.3%), total carotenoids (173.2%) and anti-collagenase activity (15.9%) compared to the maceration method. These findings provide UAE as a green and scalable method for maximizing the phenolic yield and support the application of Tetraselmis tetrahele in the development of high-value products across pharmaceutical, cosmetic, and functional food industries. Microalgae are gaining attention as promising natural sources of bioactives due to their rich nutrient content. This study investigates the effects of ultrasound-assisted extraction (UAE) parameters (i.e., solvent composition, temperature, solid-to-solvent ratio, and time) on the extraction of phenolic compounds from Tetraselmis tetrahele. The UAE parameters were determined at a solid-to-solvent ratio of 1:75, a temperature of 25 °C, and an extraction time of 15 min with 75% ethanol as the solvent. Phenolic profile, total phenolic content, total flavonoid content, radical scavenging activity of DPPH and ABTS, photosynthesis pigment (chlorophyll a, b, and total carotenoid), anti-collagenase activity, and metabolic profile of Tetraselmis tetrahele extract obtained using UAE were determined. Tetraselmis tetrahele extracts obtained using the selected UAE method demonstrated significantly higher (p < 0.05) levels in gallic acid (1.81%), 4-hydroxybenzoic acid (7.0%), total phenolic content (84.0%), total flavonoid content (56.2%), ABTS (101.3%), chlorophyll a (119.4%), chlorophyll b (174.3%), total carotenoids (173.2%) and anti-collagenase activity (15.9%) compared to the maceration method. These findings provide UAE as a green and scalable method for maximizing the phenolic yield and support the application of Tetraselmis tetrahele in the development of high-value products across pharmaceutical, cosmetic, and functional food industries.