Abstract: A DNA‐encoding thioredoxin‐carp ovarian cystatin (trx‐cystatin) was ligated into pET‐23a(+) and transformed into Escherichia coli AD494(DE3). High level of soluble recombinant trx‐cystatin, expressed in E. coli was purified by 5 min of heating at 70 °C, Q‐Sepahrose HP, and Sephacryl S‐100 HR chromatographs. Its molecular mass was 28 kDa. It could be cleaved into a recombinant thioredoxin (16 kDa) and a mature carp ovarian cystatin (12 kDa) by enterokinase. The 12‐kDa mature carp ovarian cystatin was further purified by FPLC Superdex 75 chromatography. Both recombinant trx‐fused and carp ovarian cystatins were thermostable proteins and exhibited papain‐like protease inhibition activity comparable to the wild‐type cystatin.