The invention discloses a monoclonal cell strain stably expressing a serine protease gene, a preparation method of the monoclonal cell strain, a kit containing the monoclonal cell strain, and application of the monoclonal cell strain. The monoclonal cell strain stably expressing the serine protease gene is cultured in a culture plate, and cell culture liquid is removed; classical swine fever virusraw liquid is subjected to doubling dilution, and the diluted virus liquid is added into the culture plate with the cell culture liquid being removed; and culture continues, a classical swine fever virus TCID50 is judged through a CPE phenomenon, and the TCID50 is calculated through a Reed-Muench method. The advantages that the monoclonal cell strain is used for visual detection of the classicalswine fever virus, the classical swine fever virus TCID50 is detected through the detection kit, a fluorescent microscope and a classical swine fever detection antibody are not needed, result judgement of the classical swine fever virus TCID50 is simple, and experimental repeatability is good are achieved.