The invention involves a cell line which can express human mutated tissue-type plasminogen activator (TNK-TPA), and its preparation methods. The collection number of cell lines in this invention is CCTCC C200006. We first used a DNA fragment from the short arms of human group D or group G chromosomes or its homolog to construct a recombinant human source gene vector-TNK-TPA (collection number is CCTC m200032); TNK-TPA gene is then incorporated into the target site in the nucleolus organizing region (NOR) of human group D or G chromosomes in host cell line HT1080; of which the recombinant cell line is attained after screening. The recombinant cell line can be used for manufacturing protein.