Abstract: An online in-situ multi-channel detection flow path control system using a new type of enzyme biosensing electrode prepared based on Prussian Blue (PB) nanomaterials was developed for monitoring multi-component in the microbial fermentation. According to the micro-current response characteristics of enzyme biosensing electrodes, a multi-channel pump-valve synergy flow path control system was designed to reduce cross-contamination and improve equipment reliability effectively. The software program modules such as precise positioning of pipette needles, precise micro-injection and calibration detection during multi-component detection were developed according to the logic timing requirements of flow path detection. The precise positioning of the pipette needle and precise micro-injection were realized by combining the trapezoidal acceleration and deceleration algorithm and the stepping motor controlled by subdivision drive circuit. Multi-component, wide-range, fast and accurate detection was performed by independently adjusting the injection volume and calibration detection module. The experimental test results show that the system could detect glucose, lysine and lactic acid in a wide range of concentrations, and the detection accuracy was less than 2%, which can better meet the requirements of simultaneous online detection of multiple components of microbial fermentation.