Tissue plasminogen activator (tPA) as a serine protease with 72 kD molecular mass and 527 amino acids plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The collective production of this drug in plants such as cucumber, one of the most important vegetables in the world, could reduce its production costs. In this study, after scrutiny of the appropriate regeneration of cucumber plant (Isfahan variety) on MS medium with naphthalene acetic acid hormone (NAA; 0/1 mg L⁻¹) and benzyl amino purine hormone (BAP; 3 mg L⁻¹) hormones, the cloned human tPA gene under the CaMV 35S promoter and NOS terminator into pBI121 plasmid was transferred into cotyledon explants by Agrobacterium tumefaciens strain LBA4404. Subsequent to the regeneration of inoculated explants on the selective medium, the persistence of tPA gene in recombinant plants was confirmed by polymerase chain reaction (PCR) with specific primers. To evaluate the tPA gene expression in transgenic plants, RNA was extracted and the tPA gene transcription was confirmed by reverse-transcription (RT) PCR. Followed the extraction of protein from the leaves of transgenic plants, the presence of tPA protein was confirmed by dot blot and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis in order to survey the production of recombinant tPA protein. The enzyme-linked immunosorbent assay (ELISA) test was used for recombinant tPA protein level in transgenic cucumber plants. It was counted between 0.8 and 1%, and based on this, it was concluded that the presence of three expressions of regulatory factors (CaMV 35S, Kozak, NOS) and KDEL signal in the construct caused the increase of the tPA gene expression in cucumber plants.