The invention relates to a method for rapidly obtaining a CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats/Cas9) gene knockout stable cell line through monoclonal cell sorting and belongs to the technical field of genetic engineering and genetic modification. According to the method, a CRISPR/Cas9 system, single-cell sorting by a flow cytometer and fluorescent protein screening on an expression vector are combined, positive monoclonal cells can be obtained in short time, and gene knockout work efficiency of the cell line is greatly improved. Compared with a traditional cell line gene knockout method, the flow cytometer is utilized for single-cell sorting, on one hand, tedious work of antibiotic screening is omitted, so that a large quantity of single cells can be obtained in quite short time, and on the other hand, the condition that cells in each culture hole are single cells can be guaranteed and false positive rate is reduced. Sorting is performed 40-80 h after cell transfection, at the time point, the highest survival rate of the cells after sorting can be guaranteed, and the screening efficiency is improved accordingly.