The proliferation of Sargassum biomass in various coastal areas has led to environmental and socio-economic problems. However, due to their unique composition, these biomasses offer versatile applications, prompting research into their potential in third-generation biorefineries. In this study, the hydrothermal processing of Sargassum sp. was evaluated under specific conditions at 190 °C/50 min and 150 °C/30 min. The resulting hydrolysates (liquid phase) were used as alternative culture media for cultivation. Nine treatments for the cultivation of Arthrospira platensis were assessed, varying the concentration of hydrothermal hydrolysates (HH) at 190 °C/50 min: T1 (5% v/v), T2 (10% v/v), and T3 (15% v/v). T4 (5% v/v), T5 (10% v/v), and T6 (15% v/v), maintaining the same HH conditions, and with the addition of 0.7 g/L NaNO3; and treatments T7, T8, and T9 had concentrations of 5%, 10%, and 15% of HH, respectively, at 150 °C/30 min with the addition of 0.7 g/L NaNO3, respectively. Each treatment was inoculated with 15% (v/v) of A. platensis. Growth kinetics were performed by sampling every three days for 24 days. Quantification of soluble proteins was performed for the best conditions of biomass production. The microalgae demonstrated the ability to grow under mixotrophic medium conditions and to utilize the available carbon sources in the culture medium. Treatment 4 has the highest biomass, with an Xmax (g/L) of 1.94 ± 0.06 and a protein production of 24.17 ± 0.86% (w/w). Therefore, this microalgal biomass can be used in the food matrix according to the biorefinery concept.