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House dust mites are one of the most important allergen sources worldwide and affect approximately 50% of asthmatic patients. Allergen-specific immunotherapy (AIT) is the only disease-modifying treatment for allergic diseases. However, clinical applications of allergen extract-based AIT were greatly restricted due to the potential adverse reactions. In order to improve the efficacy and reduce adverse effects, modified hypoallergens have been proposed for molecular forms of AIT. Therefore, in the present study, we converted the major house dust mite allergen Der f 34 into a B cell epitope-based hypoallergenic vaccine by the immunoinformatics and peptide-carrier fusion approaches. Initially, the physiochemical and structural properties of Der f 34 were analyzed. Accordingly, the linear and conformational B cell epitopes, as well as the helper T lymphocytes epitopes, were computed based on the properties of Der f 34. Three different fragments (residues 12–18, 83–89, and 98–116) of major allergen Der f 34 that containing candidate B cell epitope and that without T cell epitopes were linked at the N terminal and C terminal of the PreS carrier. The three-dimensional structure of the final vaccine was then predicted and the interaction with immune receptors (toll-like receptor-3) was evaluated by ligand-receptor docking. The immunogenic profiles and immune response of the final vaccine were in silico assessed after immunization, which represented the vaccine could induce an effective immune response. In addition, the codon sequences of the vaccine were cloned and expressed in E.coli, the vaccine was purified and exhibited a lower IgE-binding ability. Our results indicated that the Der f 34 hypoallergen could be a potential vaccine candidate for molecular forms of AIT in the house dust mite allergy. [ABSTRACT FROM AUTHOR]