RhNTA protein is a new thrombolytic agent which has potential medicinal and commercial value. Protein refolding is a bottleneck for large-scale production of valuable proteins expressed as inclusion bodies in Escherichia coli. The denatured rhNTA protein was refolded by an improved size-exclusion chromatography refolding process achieved by combining an increasing arginine gradient and a decreasing urea gradient (two gradients) with a size-exclusion chromatography refolding system. The refolding of denatured rhNTA protein showed that this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial rhNTA protein concentration up to 20 mg/mL. After refolding by two-gradient size-exclusion chromatography refolding processes, the refolded rhNTA was purified by ion-exchange and affinity chromatography. The purified rhNTA protein showed one band in SDS-PAGE and the specific activity of purified rhNTA protein was 110,000 U/mg. Copyright © 2008 John Wiley & Sons, Ltd.