Abstract: The invention relates to genetic engineering, and more particularly to a Dnmt3b gene-deficient CHO cell line, a preparation method and an application thereof and a recombinant protein expression system using the same. The invention adopts a CRISPR/Cas9 gene editing technique to knock out the Dnmt3b gene from the CHO cells to produce the Dnmt3b gene-deficient CHO cell line, which can significantly improve the expression level and stability of the target gene in CHO cells, overcoming the defects existing in the current CHO cell expression system, such as low expression level and stability. It has been demonstrated that using the CHO line provided herein to express a recombinant adalimumab can significantly increase the expression level of the recombinant adalimumab, indicating that the CHO cell line can be widely used to enhance the expression of target proteins.