Family 5 extracellular solute-binding protein (ESBP) is a component of the extracellular vesicle (EV) secreted by Bifidobacterium longum KACC 91563. The property of ESBP that effectively alleviates food allergy symptoms via mast cell specific apoptosis was demonstrated in our previous work, and it has revealed a therapeutic potential of this protein in allergy treatment. In the present study, B. longum KACC 91563 ESBP gene was cloned into the histidine-tagged (His-tag) pET-28a(+) vector, and the resulting plasmid was transformed into the Escherichia coli strain BL21(DE3). The transformed cells were used to grow and express the histidine-tagged ESBP and the expressed protein was purified using a Ni-NTA affinity column. To enhance the efficiency of the protein purification, three parameters were optimized; the host bacterial strain, the culturing and induction temperature, and the purification protocol. After the optimization process, two liters of transformed culture produced total of 7.15 mg of the recombinant ESBP proteins with molecular weights of approximately 60.0 kDa. This is the first study describing the production of ESBP using recombinant DNA technology. Establishment of large-scale production strategy for ESBP protein will contribute to the development a potential alternative treatment for allergies.