Tobacco industry wastewater, containing high levels of nicotine, organic carbon, dyes, and other pollutants, presents significant environmental and health challenges. This study evaluates the combined application of wood-derived biochar and microalgal biorefineries for effective treatment of this wastewater. A 3 % biochar dosage was determined to be optimal, achieving complete nicotine removal, enhancing wastewater transmittance from 12.29 % to 88.8 %, and reducing chemical oxygen demand (COD) levels by 87.7 % to 1757.3 mg/L, creating conditions favorable for the growth of Auxenochlorella pyrenoidosa. The pretreatment preserved key nutrients, such as sugars, total nitrogen (TN), and total phosphorus (TP), which supported enhanced microalgal photosynthesis and led to significant increases in biomass and lipid accumulation, particularly in unsaturated fatty acids. This shift in lipid composition contributed to a significant reduction in the cold filter plugging point value, improving the suitability of microalgae-derived biodiesel for use in cold climates. Transcriptomic analysis revealed upregulation of pathways involved in photosynthesis, carbon fixation, and fatty acid biosynthesis, while downregulation of the tricarboxylic acid (TCA) cycle minimized acetyl-CoA consumption, further promoting lipid synthesis. This integrated approach not only effectively detoxifies tobacco industry wastewater but also converts it into valuable biofuels, offering a promising and sustainable strategy for waste management and biofuel production.