Abstract: The fermentation process of β-mannanase produced by Bacillus licheniformis TJ-101 was analyzed in a 6.6-L fermenter. The internal relationship between the operation parameters and the online testing parameters was also elucidated during the fermentation process. The results showed that the key factors, including rotate speed, aeration rate and fermentation time, had an important effect on the β-mannanase production. Furthermore, the above three fermentation parameters for producing β-mannanase by Bacillus licheniformis TJ-101 were optimized via response surface methodology (RSM) . The optimum conditions were rotate speed of 646.5r/min, aeration rate of 5.0L/min and fermentation time of 45.28 h, respectively. Under these conditions, the maximum activity of β-mannanase was 440.52U/m L. Compared with the value before optimization, the activity was increased by 44.5%.