In order to study the effect of online monitoring fed-batch fermentation of living cells on L-hydroxyproline fermentation, the live cell online monitor was used in the 30 L fermentor to monitor the number of living cells to adjust the feeding strategy, and the effect of online monitoring fed-batch fermentation of living cells on sugar supplement rate, glucose conversion rate and the metabolic flux channeled were analyzed.The results showed that the best sugar supplementation strategy for the production of L-hydroxyproline by the online monitoring of the living cells was to reduce the rate of sugar supplementation timely in the mid and later phase of fermentation, and finally to 9 g/(L·h).The production of L-hydroxyproline increased by 11.39%, the accumulation of acetic acid decreased by 48.69%, and glucose conversion rate increased by 14.92%.At the same time, the flux channeled to EMP and glyoxyl circle decreased by 8.50% and 16.89%, respectively.However, the flux channeled to HMP increased by 14.73%.The flux channeled to acetic acid decreased by 4.97%, and the metabolic flux channeled to L-hydroxyproline increased by 16.21%.These results indicated that online monitoring fed-batch fermentation of living cells could significantly reduce the accumulation of by-product, increase the production of L-hydroxyproline, and improve the glucose conversion rate.