Oxygen supply and oxygen uptate rate (OUR) significantly influences both the physiological state of cells and nemadectin biosynthesis by Streptomyces cyaneogriseus ssp. noncyanogenus. In this study, a real-time monitoring approach for nemadectin fermentation was developed for the first time by an on-line OUR control strategy. The effect of OUR levels during the mycelium differentiation phase on nemadectin biosynthesis was studied and optimized by adjusting feeding rates of (NH₄)₂SO₄. Results showed that controlling the OUR at approximately 17.5 mmol·L^-1·h^-1 during the mycelium differentiation phase effectively enhanced nemadectin production, reaching 2805.1 μg/mL, making 129.5% increase compared to the control. This OUR control strategy was successfully scaled-up to a 500 L pilot scale, achieving the highest ever nemadectin production of 2867.3 μg/mL. These findings demonstrate that this OUR control strategy provides an effective and scalable approach for industrial nemadectin production.