The invention discloses a preparation method of a cell strain capable of expressing glucocerebrosidase with high mannose content. The preparation method comprises the following steps: (1) carrying outlipofection transfection, namely, aiming at the sequence of a GNT1 gene, designing a sgRNA sequence guiding cutting of endonuclease, recombining the sgRNA sequence into a knock-out vector, thus obtaining synthetic plasmids with coexpression of sgRNA and endonuclease, and transfecting a cell pool stably expressing glucocerebrosidase by adopting the synthetic plasmids through a liposome transfection method; (2) carrying out cloning by adopting a limiting dilution method, namely, screening out monoclonal cells, and carrying out enlarged culturing; (3) carrying out mutation cloning screening on the target gene, namely, acquiring monoclone with the GNT1 gene knocked out through cell PCR sequencing, and thus the cell strain capable of expressing glucocerebrosidase with high mannose content is obtained. The cell strain can stably express glucocerebrosidase, meanwhile, the content of the mannose is obviously improved, the binding capacity with mannose receptor is effectively improved, the enzyme activity capacity is improved, and the purpose of increasing the efficacies is achieved.