The invention discloses a microfluidic device and method for optimal control on microbial fertilizer bacterium culture conditions. The device comprises a case body and a PC (personal computer) positioned outside the case body, wherein a gas pump, a 10-channel constant-pressure sampler, an objective table, a microcontroller and a three-stage microfluidic chip are fixedly arranged in the case body. A complete optimal control method of nutrient solution main component screening, main component concentration proportion optimization and sample introduction parameter and temperature optimization of the optimal proportion nutrient solution is adopted to implement the microfluid intelligent control, temperature control and intelligent monitoring double feedback and also implement microfluid intelligent control, external shape image automatic treatment and interior physiologic metabolite activity electrochemical detection double feedback, thereby directly controlling the multichannel sample introduction mode and sample introduction parameters to perform the microfluidic microbial fertilizer bacterium culture. The method can be used for culture condition optimal control on any microbial fertilizer bacterium under the condition of unknown nutrient solution components.