Expression efficacy of recombinant protein in current expression systems is generally low. Therefore， the expression levels of recombinant proteins in the breast milk of transgenic animals are typically low. In view of this， the present study aimed to construct homozygous transgenic rabbits with a high expression level of recombinant human plasminogen activator (rhPA) during the entire lactation period. Homozygous transgenic rabbits were obtained using an effective rhPA mammary‑specific expression vector PCL25/rhPA. The expression level and thrombolytic ability of rhPA in the milk of both homozygous and hemizygous rabbits were detected by enzyme‑linked immunosorbent and fibrin agarose plate assays. It was observed that the expression of rhPA was constant during the entire lactation period in homozygous rabbits， while the expression of rhPA declined slowly in hemizygote rhPA transgenic rabbits during the lactation period. In addition， the expression of rhPA in homozygous transgenic rabbit was ~950 µg/ml， which was markedly higher in comparison with that in hemizygote rabbits. Furthermore， increased gene copy number was observed to increase the expression level of rhPA at the same integration vector.