OBJECTIVE To establish SEC-HPLC method for purity determination of recombinant human TNK tissue type plasminogen activator (rhTNK-tPA).METHODS Monomer and aggregate of rhTNK-tPA were determined by SEC-HPLC method directly; For the anal. of double-chain, disulfide bond of the samples were first opened by dithiothreitol (DTT), and then the HPLC-SEC method was adopted.The column was TSKgel G3000SWXL, and the mobile phase was 3%NaH2PO4-0.1%SDS (pH 6.8); The flow rate was 0.3 mL·min-1, the detection wavelength was set at 214 nm, and the column temperature was 25°C.The detection time was 50 min.RESULTS The specificity and precision of the tests were very good, the Monomer, Aggregate and double chain content of rhTNK-tPA (three batches of bulk) were (98.8%, 1.2%, 8.0%), (98.9%, 1.1%, 7.5%), (99.0%, 1.0%, 7.5%), resp.CONCLUSION The method is accurate and comprehensive, it can be used for the quality control of rhTNK-tPA at higher level.