Introduction: Leishmania tarentolae (L. tarentolae) is a nonpathogenic species of Leishmania genus that can be used as an expression system to produce immunogenic proteins or epitopes in vivo acting as an efficient and safe recombinant live vector in vaccinology. Cysteine proteases (CPs) are one group of Family C1 peptidases in Leishmania that are important for survival of the parasite within the host and are introduced as potential vaccine candidates. Two types of cysteine proteases, CPA and CPB, play a critical role in Leishmania. Previously, it has indicated that immunization with two genes or recombinant proteins of CPA and CPB individually or fused together with various adjuvant or combined with liposome are able to elicit a protective immune response against L. major in BALB/c mice. Methods: In this study, for the first time, two lines of recombinant non-pathogenic Leishmania were generated by transfection of a heterologous triple fusion gene encoding cpa/cpb/egfp. For this purpose, two different expressions approaches were taken episomal expression using rDNA promoter, and integrative expression from rRNA locus of genome. Results: After genotype confirmation, the fluorescence intensity was monitored in different phases of parasite growth by both fluorescence microscopy and FACS analysis.Western blot and RT-PCR analysis showed that cpa/cpb/egfp tri-fused genes were specifically expressed in both lines of transgenic parasites. Discussion: In this study a single fused gene was expressed in both systems and the results showed that the expression of integrated gene was higher than episomal. In addition, the present data suggest that L. tarentolae expressing cpa/cpb/egfp is a promising carrier as vectored vaccine in future research. Vac Res , 2014, 1 (1): 1-9