Clostridium botulinum is considered the most commonly reported bacterium that causes food poisoning. It has a huge impact on the health of people around the world. Given the pressing nature of the situation, it is crucial to expedite the development of a vaccine to effectively counter it. Most experimental work without bioinformatics preliminary work is time-consuming. Clostridium botulinum is a type of Gram-positive bacterium that can form spores and is considered a leading cause of food poisoning related to antibiotic use. The prevalence of infections caused by this bacterium has increased significantly over time, resulting in more hospitalizations and deaths. When the spores are ingested, they can germinate in the gastrointestinal tract, causing contamination and resulting in an infection. The bacterium's ability to colonize surfaces is attributed to surface layer proteins and flagellar proteins, while sporophyte proteins facilitate spore invasion. The recurrence of Clostridium botulinum infection in hospitalized patients is mainly attributed to two factors. While previous studies have focused on specific proteins, this study aims to analyze the entire proteome to identify more immunogenic proteins. Using immunoinformatics, a chimeric vaccine candidate is formed by selecting proteins to create a multivalent epitope. To ensure the fidelity of our vaccine candidates, we performed stability tests using in silico and molecular dynamics simulations. Additionally, docking studies were performed to confirm the stable interaction of the vaccine with Toll-Like receptors and MHC receptors present in innate immune cells. In-silico codon optimization was also carried out to optimize the vaccine, which was then introduced into a cloning vector. The efficacy of the vaccine was evaluated by examining its expression in both Homo sapiens and E. coli expression systems. The efficacy of vaccine candidates in inducing defensive immune responses was evaluated using an in silico immune simulation system. Our study has successfully generated promising short-peptide vaccine candidates against Clostridium botulinum using computational tools, which could potentially save time and resources. These findings provide valuable information for future in vitro and in vivo studies on this topic