The present invention provides a method of producing an antibody-producing hybridoma, which includes fusing a B cell immunized in vitro and a myeloma cell, and which is more efficient and practical than the conventional methods, more specifically a method of producing an antibody-producing hybridoma, which includes fusing a B cell immunized with an antigenic substance in the coexistence of a cytokine and a glycolipid in vitro, and a myeloma cell, and use thereof.