This study investigates the sustainable production of fatty acids, specifically docosahexaenoic acid (DHA omega-3), which is essential for the nutrition, cosmetics, and pharmaceutical sectors. The research investigation evaluates the potential of utilizing low-cost substrates in a circular economy framework, employing Aurantiochytrium microalgae, a species recognized for its elevated DHA content and lack of heavy metal contamination. The cultivation process employed three substrates: glycerol, molasses, and fruit waste. The microalgae were cultivated on these substrates, subsequently undergoing sonication to improve emulsion stability. Fatty acid profiles were analyzed using GC-MS to assess DHA yields and the efficiency of biomass production. The findings suggested that glycerol served as the most effective substrate, producing the highest DHA content (54.88%) and wet biomass (53 g). Molasses and fruit waste exhibited moderate efficiency, presenting viable and cost-effective alternatives. Furthermore, glycerol yielded the most uniform emulsion particles (1,874 nm, PI 0.02677), suggesting enhanced substrate compatibility. The findings highlight the capability of Aurantiochytrium-based bioprocesses for the sustainable production of high-value fatty acids. This approach leverages industrial and organic waste materials, enhancing environmental sustainability and economic viability while fostering innovation in microalgae biotechnology.