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Entamoeba histolytica , an anaerobic parasite, infects humans and other primates and causes fatal diseases, such as amebiasis, amebic liver abscesses, and many others. Thousands of people are infected and dying due to the need for a proper protective cure, especially in poor sanitizing regions, such as Latin America, Asia, and Africa. Around 10% of the world population is infected by E. histolytica every year. Consequently, novel preventive approaches are required to eliminate the threats of the parasite. A designed vaccine targeting the exposed proteins that are common between cyst and trophozoite stages of the parasite's life cycle would be an effective way to repress the impact of the parasite. Therefore, an in silico bioinformatics approach was performed to design an effective vaccine targeting surface proteins common between both stages of the parasite's life cycle using B-cell and T-cell epitopes. The epitopes derived from the conserved portions of the proteins and their corresponding isomers specific to the parasite suggested that the vaccine could benefit cross-protection. Furthermore, the three-dimensional structure of the designed vaccine was modelled, refined, and validated using multiple bioinformatics tools. The physiological properties and solubility were also predicted using different algorithmic tools and found to be highly soluble in nature. The vaccine was found interactcted with TLR immune receptors, and the stability was observed via dynamics simulation. Codon optimization and cloning were performed for expression analysis. Immune simulation prediction anticipated significant immune responses with a high IgG and IgM antibodies expression, Th and Tc cells population, B-cell population, memory cells, INF-γ, and IL-2 cytokines. Therefore, the constructed multi-epitope putative vaccine can effectively neutralize the parasite's harmful effects. • A novel chimeric vaccine was made using epitopes originating from 8 proteins expressed in the cyst and trophozoite stages of Entamoeba histolytica. • The designed vaccine showed significant interactions with toll-like receptor 2 and toll-like receptor 4 by docking analysis and dynamics simulation. • The vaccine showed to induce a robust immune response observed by the immune simulation. [ABSTRACT FROM AUTHOR]