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The <i>Agrobacterium</i>-mediated transient gene expression system is a rapid and efficient method for heterologous recombinant protein expression in plants. The fermentation of genetically modified <i>Agrobacterium tumefaciens</i> is an important step in increasing the efficiency of recombinant protein production in plants. However, the limitation of this system that makes it economically non-competitive for industrial-scale applications is the <i>Agrobacterium</i> suspension production cost. In this study, the utilization of sugarcane molasses as an alternative low-cost source of carbon at a concentration of 8.7 g/L and nitrogen at a concentration of 2.4 g/L for <i>Agrobacterium</i> cultivation was investigated. Molasses pretreatment using sulfuric acid (SA) was applied before fermentation, and it resulted in a maximum specific growth rate of 0.232 ± 0.0063 h⁻¹ in the <i>A. tumefaciens</i> EHA105 culture. The supplementation of antibiotics in the molasses-based medium was shown to be unnecessary for plasmid maintenance during fermentation in both <i>Agrobacterium</i> strains, which helped to reduce the production cost. We evaluated recombinant protein production using an <i>Agrobacterium</i> culture without antibiotic supplementation in the growth media by demonstrating green fluorescent protein expression in wild-type <i>Nicotiana benthamiana</i> leaves. In the evaluation of the culture medium cost, the molasses-based medium cost was 6.1 times lower than that of LB. Finally, this study demonstrated that the newly developed molasses-based medium for <i>Agrobacterium</i> fermentation is a feasible and effective medium for transient recombinant protein production in plant tissues.