α-L-Rhamnosidase was an important hydrolytic enzyme in industry.We had investigated the relation between the mycelium viability and the yield of α-L-rhamnosidase to optimize and monitor the enzyme production in 5 L fermentation tank.The mycelium viability was measured by the triphenyltetrazolium chloride(TTC) method.The results showed that maintaining appropriate mycelium viability in fermentation was significant for α-L-rhamnosidase production.When the mycelium viability of inoculum in shake flask reached to the maximum 0.388 U/mL,the yield of α-L-rhamnosidase in 5 L fermentation tank was the highest.Controlling the pH at 5.5 during fermentation could effectively slow down the decline speed of mycelium viability in fermentation end,and correspondingly increase 28.7% of the α-L-rhamnosidase yield.When the agitation speed was below 400 rpm/min or the air flow was less than 0.3 vvm,the maximum mycelium viability and α-L-rhamnosidase yield was remarkably low.Therefore,the mycelium viability could be used as an important parameter in fermentation optimization and process control of α-L-rhamnosidase production.