Fungi use different tools to overcome plant defense mechanisms. Phytoanticipins are a part of defense materials which are produced in plants to prevent invasion of pathogens. Tomatinase is produced and secreted by a range of fungi (including some forma specialis of Fusarium oxysporum ) to degrade phytoanticipins. In this study due to agricultural importance of melon crop in the Khorasan provinces, Tomatinase presence have been investigated in genome of race 1 and 1.2 of F. oxysporum f. sp. melonis causal agent of Fusarium wilt disease as an economically important disease of melon. For this purpose, specific primers PSh30-F/R based on the F. oxysporum f. sp. lycopersici Tomatinase gene were designed after defining the specific sites in up- and down-stream of the gene. Result revealed the specific primer pair amplified a single 1.1 kb band of the expected size in all tested isolates. The segment cloned in pTG19-T vector and transferred to Dh5?. Recombinant colonies confirmed by colony PCR technique using the specific primer pair and M13 universal primers. A confirmed colony selected for sequencing and Sequencing results showed some variations in Tomatinase sequence of FOM. To design an expression construct of Tomatinase , the specific primers Psh30.3-F/R based on Spe 1 and Sac 1 restriction site were designed. Results showed the specific primer pair amplified single 1056 bp band of Tomatinase carrying the Spe 1 and Sac 1 sites. To express the protein, amplified Tomatinase ligated to pET vector and transferred to BL21. This construct (pETFOM) was sequenced and results revealed that pETFOM construct was designed in correct frame and it doesn’t have any stop codon. Expression of Tomatinase protein required additional factors such as bacterial strain changing, reduction of induction temperature and the other concentration of IPTG. The expression of Tomatinase was confirmed with RT-PCR. So the results based on multiple alignment of Tomatinase showed that FOMTom is a member of glycosyl hydrolases family 10 and is closed to FoTom1 . Keywords: melon, fusarium wilt disease, Tomatinase, gene detection.