The present invention relates to a novel method for isolating and purifying tissue plasminogen activator and its variants, and more specifically TNK-tPA from CHO cells, which is industrially applicable, simple and cost effective. A high, robust and highly efficient TNK-tPA purification method is described.本発明は、組織プラスミノーゲン活性化因子及びその変異体、より具体的にはTNK-tPAをCHO細胞から単離及び精製する新規方法に関し、工業的に適用可能で、簡単で、費用対効果が高く、堅牢で、高効率なTNK-tPA精製方法を記載する。