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.