The human tissue plasminogen activator (t-PA) is a thrombolytic protein that plays a key role in fibrinolysis by converting the plasminogen into plasmin which degrades fibrin clots in blood vessels. The t-PA gene encoding the human tissue plasminogen activator was cloned and expressed under transcriptional control of single and multiple rooting loci promoter (rolD) promoters in hairy roots derived from Oriental melon. t-PA gene integration in the genome of hairy roots was confirmed by PCR. Increased transcript and protein expression levels was observed in the transgenic hairy roots expressing t-PA under the control of single and dual rolD promoters, respectively. Enzyme-linked immunosorbent assay revealed the highest levels of the recombinant t-PA accumulation in transgenic hairy roots carrying the t-PA transgene under the control of single and dual rolD (0.17-0.15 μg/mg) promoters as compared to triple and quadruple rolD (0.07-0.03 μg/mg) promoters. Fibrinolytic activity indicated the proper function of the expressed recombinant t-PA in fibrin lysis. These results demonstrated that hairy roots could serve as alternatives to transgenic plants for expression of mammalian genes.