Arthrospira platensis holds promise for biotechnological applications due to its rapid growth and ability to produce valuable bioactive compounds like phycocyanin (PC). This study explores the impact of salinity and brewery wastewater (BWW) on the mixotrophic cultivation of A. platensis. Utilizing BWW as an organic carbon source and seawater (SW) for salt stress, we aim to optimize PC production and biomass composition. Under mixotrophic conditions with 2% BWW and SW, A. platensis showed enhanced biomass productivity, reaching a maximum of 3.70 g L−1 and significant increases in PC concentration. This study also observed changes in biochemical composition, with elevated protein and carbohydrate levels under salt stress that mimics the use of seawater. Mixotrophic cultivation with BWW and SW also influenced the FAME profile, enhancing the content of C16:0 and C18:1 FAMES. The purity (EP of 1.15) and yield (100 mg g−1) of PC were notably higher in mixotrophic cultures, indicating the potential for commercial applications in food, cosmetics, and pharmaceuticals. This research underscores the benefits of integrating the use of saline water with waste valorization in microalgae cultivation, promoting sustainability and economic efficiency in biotechnological processes.