Glioblastoma (GB) is a common primary malignancy of the central nervous system, and one of the highly lethal brain tumors. GB cells can promote therapeutic resistance and tumor angiogenesis. The CD171 is an adhesion molecule in neuronal cells that is expressed in glioma cells; it is a regulator of the brain development. CD171 is one of the immunoglobulin-like CAMs (cell adhesion molecules) families that can be associated with prognosis in a variety of human tumors. The multi-epitope peptide vaccines are based on synthetic peptides with a combination of both B-cell epitopes and T-cell epitopes, which can induce specific humoral or cellular immune responses. In the present study, several immune-informatics tools were used for analyzing the CD171 sequence and studying the important characteristics of a designed vaccine. The results included the prediction and validation of the secondary and tertiary structure, physicochemical properties, solubility, conservancy, toxicity as well as antigenicity and allergenicity of the promising candidate for a vaccine against CD171. The immunoinformatic analysis suggested 12 predicted multi-epitope peptides, whose construction consists of 582 residues long. Therewith, cloning adaptation of designed vaccine was performed and eventually sequence was inserted into pET30a (+) vector for the application of the anti-glioblastoma vaccine development.