Epstein-Barr virus (EBV) is a global lymphotropic virus and has been associated with various malignancies, among which colorectal cancer (CRC) is the prevalent one causing mortality worldwide. In the recent past, numerous research efforts have been made to develop a potential vaccine against this virus; however, none is effective possibly due to their low throughput, laboriousness, and lack of sensitivity. In this study, we designed a multi-epitope subunit vaccine that targets latent membrane protein (LMP-2B) of EBV using pan-genome and reverse vaccinology approaches. Twenty-three major histocompatibility complex (MHC) epitopes (five class-I and eighteen class-II) and eight B-cell epitopes, which have been found to be antigenic, immunogenic, and non-toxic, were selected for the vaccine construction. Furthermore, 24 vaccine constructs (VCs) were designed from the predicted epitopes and out of which VC1 was selected and finalized based on its structural parameters. The functionality of VC1 was validated through molecular docking with different immune receptors (MHC class-I, MHC class-II, and TLRs). The binding affinity, molecular and immune simulation revealed that the VC1 had more stable interaction and is believed to elicit good immune responses against EBV. HIGHLIGHTS: Pan-genome and reverse vaccinology approaches were used to design a multi-epitope subunit vaccine against LMP-2B protein of EBV. Epitopes were selected based on the antigenic, immunogenic, and non-toxic properties. Twenty-four vaccine constructs (VCs) were designed from the predicted epitopes. Designed vaccine VC1 has shown good binding affinity and molecular and immune simulation. VC1 was validated using molecular docking with different immune receptors.