In recent years, using plants as bioreactors for producing valuable recombinant peptides have gained a dominant part of the researches, because of their advantages like product safety, low costs and post translational modifications. One of the valuable peptides which its high production costs, have led to considering other recombinant protein production systems like plants for cheaper and safer production is tissue plasminogen activator (tPA). Glycoprotein tPA is a highly priced medicine which is specifically used for solving blood coagulations caused by some heart and vein diseases like ischaemic stroke and acute myocardial infarction. The aim of the current study is to evaluate the production capability of recombinant tPA in plant system using tobacco and lettuce hosts and also comparing the impact of Zera, Extensin and KDEL signal peptides on tPA recombinant protein production in plant systems. In this study, cDNA of tPA gene under the control of CaMv 35S promoter and NOS terminator in companion with three Zera, Extensin and KDEL signal peptides in forms of three different constracts named pZt, pExt and pKt was transferred into tobacco and lettuce nuclear genomes by Agrobacterium mediated- gene transfer technique and the presence of all the constructs in T 0 and T 1 transgenic plants were confirmed through PCR and RT-PCR techniques. The results obtained from the Eliza test on transgenic tobacco plants showed that an increase in tPA recombinant protein production in T 0 transgenic plants but it was low in transgenic plants with pKt and pExt, and very low for pZt constructs respectively. There was also no significant difference between non-transgenic plants and transgenic ones. Data analysis results from the production of tPA recombinant protein in T 1 transgenic plants containing pZt, pExt and pKt constructs were completely different and were 0.03, 0.014 and 0.005 nanogram per milliliter respectively. Although, in terms of producing recombinant protein, some non-uniformity were observed in T0 ones the T1 transgenic plant were used as the base caculation of signal peptide impacts. It could be concluded that despite the capability of stable transformation of tPA gene to tobacco and lettuce plants, the Zera and Extensin signal peptides are suitable construscts for producing tPA recombinant protein as compared to KDEL signal.