ABSTRACT This study investigated the effect of ultrasound-assisted enzymatic extraction (UAEE) conditions: solid-to-water ratio (1:50–1:100), ultrasonic power (296–506 W), and temperature (40–60°C) on key criteria (responses) of Gracilaria fisheri agar. Response surface methodology resulted in accurate quadratic models for predicting responses. Non-linear (quadratic and interaction) effects of UAEE conditions significantly (p < 0.05) influenced the responses. Optimal conditions (solid-to-water ratio of 1:98, ultrasonic power of 506 W, and temperature of 60°C) produced 32.02% agar yield, 58.05% DPPH antioxidant activity, and 83.48 g/cm2 gel strength, aligning with predictions. This research establishes an efficient agar extraction method with controllable qualities from red seaweed.