Abstract: Undaria pinnatifida, a marine macroalga rich in bioactive compounds, has attracted increasing attention for its mycosporine-like amino acids (MAAs), which exhibit unique UV-absorbing, antioxidant, and anti-inflammatory properties. To establish a sustainable vegetative propagation system for U. pinnatifida gametophytes and enhance MAAs accumulation, this study systematically investigated the effects of key culture conditions—such as light quality, light intensity, photoperiod, temperature, and nutrient availability—on gametophyte growth and MAAs biosynthesis using a combination of single-factor and uniform design experiments. The antioxidant activity of MAAs extract was evaluated in vitro via DPPH and ABTS⁺ radical scavenging assays, while their anti-inflammatory potential was assessed in vivo using a zebrafish inflammation model. Results indicated that culture conditions significantly affected both gametophyte growth and MAAs accumulation. Optimal conditions—red light, 20 ℃, a light intensity of 32 μmol·m⁻²·s⁻¹, and a 12L:12D photoperiod—yielded the highest relative growth rate (6.41%±0.11%) and MAAs content (327.19±1.59 mg/g). UV-visible spectroscopy confirmed the characteristic absorption profiles of MAAs in the extracts. The MAAs extract exhibited IC₅₀ values of 12.04 mg/mL and 10.34 mg/mL against DPPH and ABTS⁺ radicals, respectively. Furthermore, at 100 μg/mL, the extract reduced macrophage aggregation in zebrafish by 52.31%, demonstrating a clear dose-dependent anti-inflammatory effect. In summary, this work identifies optimal culture conditions for enhancing both gametophyte growth and MAAs production, and confirms that gametophyte-derived MAAs possess moderate antioxidant and notable anti-inflammatory activities, thereby providing a theoretical and experimental foundation for their efficient biosynthesis and potential application.