The characteristic of Bacillus subtilis fermentation process of guanosine on 50 L fermentor was analyzed. Based on determination of on-line and off-line parameter, using correlation analysis, the technology study of physiologic regulation was combined with the metabolic flux distribution of synthesis process. The metabolic flux shift from HMP to EMP and TCA cycle during fermentation was found. The reason of the flux shift was preliminary analyzed, based on which the procedure was optimized to increase the yield of guanosine to 30 g/L.