The demand for high-quality and cost-effective raw materials is growing across various industries. Canola meal (CM), a low-cost by-product of canola processing facilities, boasts an impressive nutritional profile comprising protein, carbohydrates, vitamins, and minerals, making it a potential candidate for microbial culture media. Saskatchewan leads globally in canola production and plans to expand its canola processing facilities. Commercial valorization of CM could not only optimize the management of this by-product but also offer additional incentives and financial returns to regional canola stakeholders. Efficient utilization of canola meal extract (CME) in producing polyunsaturated fatty acids (PUFAs), such as arachidonic acid (ARA), holds promise as valuable ingredients or base chemicals for the food, feed, and nutraceutical industries. This study focused on incorporating pH 10.5 treated alkaline extract (AE) and temperature 160°C treated subcritical extract (SE) of CM into the culture medium of Mortierella alpina, an oleaginous fungus, by replacing conventional yeast extract (YE). Trials investigated the effect of aeration levels ranging from 0.5 to 2.0 vvm and revealed that increasing oxygen supply significantly boosted fungal growth rate and ARA content in biomass up to 0.39 day⁻¹ and 21.86 %, respectively. ARA production correlated positively with biomass accumulation and lipid content. Replacing YE with CME resulted in less significant differences (P < 0.05) in biomass and lipid accumulation. Additionally, AE- and SE-supplemented media exhibited higher ARA accumulation of 144.91 mg/L and 165.8 mg/L, respectively, compared to standard media (124.87 mg/L). Therefore, this study introduces canola meal extract (CME) as a novel nitrogen source in microbial media, with comparative demonstrations showing its efficiency to be similar to that of conventional extracts.