This study explores the potential of Chlorella sp. BF03 isolated from a betta fish farm is a promising candidate for sustainable biomass and polyunsaturated fatty acid (PUFA) production when cultivated in palm oil mill effluent (POME). The investigation reveals substantial improvements in both biomass quantity and PUFA content under specific cultivation conditions. The most favorable outcomes were achieved when Chlorella sp. BF03 was grown in POME at a dilution ratio of 1:3, resulting in the highest biomass yield of 153.03±36.16 mg/L. Gas chromatography-mass spectrometry (GC-MS) was employed for the analysis of the fatty acid profile. The results revealed the presence of α-linolenic acid (ALA). To optimize fatty acid production, Chlorella sp. BF03 was subjected to dark stress. The findings present compelling evidence of enhanced polyunsaturated fatty acid (PUFA) content, emphasizing the potential of Chlorella sp. BF03 for sustainable PUFA production using palm oil mill effluent (POME) as a substrate.