Viral myocarditis is a moderate disease, but it sometimes causes progressive cardiac disorder. Many different viruses have been considered as the agent of viral myocarditis, but Coxsackievirus of the B group, in particular of the Coxsackievirus B3 (CVB3), is more than fifty percent of cases of viral myocarditis. CVB3 is a positive single-stranded RNA virus and a member of the genus Enterovirus and it is most commonly causing of human viral myocarditis or human acute, especially in young patients. The goal of this study is a comparison of three molecular methods included RT-PCR, NASBA and RT-LAMP for detection of CVB3. For this purpose, the primer explorer V4 software was used for designing of specific primers. Total RNA extracted from CVB3-infected HeLa cell line after 24 h and stored in-80 ° C since using as the template in RT-LAMP, NASBA and RT-PCR assays. Then, for evaluated of the sensitivity of these methods, serial dilution of total RNA was performed. The result of this study showed that the sensitivity of RT-LAMP, NASBA and RT-PCR were 0. 1, 10 and 10 pg, respectively. Based on the results that obtained in this study, the RT-LAMP assay was highest sensitive than RT-PCR and NASBA techniques for detection of CVB3 infection.