Compare with transient expression, stable cell lines generally have higher productivity and better quality for protein expression. However, selection of stable cell line is time-consuming and laborious. Here we describe an optimized selection method to achieve high-efficient stable cell pools with Expi293F suspension cells. This method only takes two to three weeks to generate stable cell pools with 2- to 10-fold higher productivity than transient gene expression (TGE). In fed-batch culture with Yeastolate, > 1 g/L yield was achieved with our KTN0239-IgG stable cell pool in shaker flasks. This method can be also applied to efficiently display proteins on the cell surface.