Glucagon-like peptide 1 (GLP-1) is an insulinotropic protein. It was reported that the continuous infusion of GLP-1 normalized the blood glucose level in type 2 diabetes animal model. However, the short half-life of GLP-1 has limited its application in clinical settings and prompted us to develop a GLP-1 gene therapy system. Our previous results showed that the delivery of pbeta-GLP-1 using polyethylenimine (PEI) reduced the blood glucose level effectively. However, the glucose level was not completely normalized. In the present study, the more efficient GLP-1 expression system was developed using two-step transcription amplification (TSTA). To evaluate the TSTA system, pbeta-Gal4-p65 and pUAS-Luc were constructed. The pUAS-Luc/pbeta-Gal4-p65 system showed the highest transfection efficiency at a 2:1 pUAS-Luc/pbeta-Gal4-p65 weight ratio. In addition, the transgene expression by the TSTA system was at least 4 times higher than pbeta-Luc. To apply the TSTA system to the GLP-1 expression plasmid, pUAS-GLP-1 was constructed. The pUAS-GLP-1/pbeta-Gal4-p65 system showed higher mRNA level than pbeta-GLP-1. In addition, the level of GLP-1 by the pUAS-GLP-1/pbeta-Gal4-p65 system was more than 4 times higher than pbeta-GLP-1. Therefore, the TSTA GLP-1 expression system may be useful to develop gene therapy system for type 2 diabetes.