The invention discloses a pilot fermentation process for expressing recombinant acetylcholinesterase (AChE) in pichia methanolica. The process is characterized by firstly culturing an activated recombinant pichia pastoris strain, thus preparing a fermentation working seed liquid, then transferring the fermentation working seed liquid to a basal salt culture medium, regulating the pH value of a fermentation system by utilizing ammonia water or phosphoric acid, then carrying out inoculation, adding proper amount of trace element solution, adding a mixed solution of glycerin and trace elements after growing to a certain stage, finally feeding a methanol solution containing trace elements to carry out protein induced fermentation, continuously maintaining the temperature at 29 DEG C and maintaining DO more than 20% in the fermentation process and carrying out induction with methanol for 120 hours, thus finishing the whole fermentation process. The pilot fermentation process has the advantages that the fermentation process is simple and is convenient to operate; the proteins are subjected to soluble expression and the expression quantities are high and the enzymatic activity is high but the protein expression cost is relatively low; large scale fermentation tank expression is carried out on AChE in a yeast expression system for the first time and lays the foundation for large scale production and final application of AChE.