The cholera toxin B subunit (CTB), a nontoxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. A gene encoding CTB, which was modified based on the optimized codon usage in the plant, was synthesized and fused to the endoplasmic reticulum retention signal KDEL to enhance its expression level in plants. The synthetic CTB (sCTB) gene was introduced into a plant expression vector adjacent to the CaMV 35S promoter, and was transformed into tomato using an Agrobacterium-mediated transformation method. The integration of the sCTB gene into the genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification. The synthesis and assembly of CTB protein in transgenic plants was demonstrated through immunoblot analysis and $G_{M1}$-ELISA. The highest amount of CTB protein produced in transgenic tomatoes was approximately 0.9% of total soluble fruit protein which was 10-fold greater than the previously 0.081%. $G_{M1}$-ELISA indicated that plant-synthesized CTB protein bound specifically to $G_{M1}$-gangliosides, suggesting that the CTB subunits formed active pentamers.