Abstract: Riboflavin plays a crucial role in metabolic pathway. Commercial riboflavin has been widely applied in a variety of different industries, such as, pharmaceuticals, cosmetics, human and animal nutrition etc. Recently, riboflavin product is mainly derived from biological process in industrial production. However, the focus of research has shifted to molecular modification of production strain genes, where very few attention is paid to the analysis of metabolic flux regulation during metabolism, with nitrogen regulation in particular. This work is aimed to explore how the interaction of inorganic nitrogen sources affects the riboflavin yield and cell density, and how the ammonium ion concentration influences the whole process in fed-batch culture. The results show that ammonium is effective in promoting the biomass but exerts an inhibitory effect on the yield. On the contrary, nitrate is beneficial to improve the output of riboflavin but has some adverse effect on biomass. In this work, ammonium and nitrate were employed with mole ratio 1∶8. The riboflavin yield was observed to reach 812 IU/mL, a 30% increase as compared to that with only use of ammonium under the identical condition. In fed-batch culture, ammonia is frequently involved in the regulation of pH to ensure nitrogen supply. Nevertheless, a careful control over its concentration is required. In 7 L bioreactor, glucose concentration was in the range of 5—10 g/L and the concentration of ammonium was controlled to below 10 mmol/L in the late fermentation. As a result, DCW was 74.5 g/L and the riboflavin yield was increased to 5 292 IU/mL, which was 10% higher than the control.