Plant viruses cause serious disease of crop plants reducing both quality and quantity of final produce. Serological tests are used all over the world in laboratory and field based detection of plant viruses and they are of great indispensable importance in agricultural production; virus certification programs; agricultural quarantine and production of virus-free crops grown for processing or fresh market. Cross reaction between viruses and their strains antisera limits serological differentiation of viruses and their strains by enzyme-linked immunosorbent assay (ELISA). This study aims to characterize the antigenic property of Tomato mosaic virus ToMV coat protein by using some bioinformatic tools to analyze its gene. Therefore, new methods in antibody production could be used as equivalent to Mabs in its high specificity. ToMV isolate was confirmed by Transmission electron microscope and differential hosts and propagated on N. tabacum cv. Samson. Systemic infection was developed on N. tabacum cv. Samson and local infection on Datora metel; D. stramonium; N. glutinosa; Chenopodium amaranticolor; C. quinoa. ToMV was purified and used as immunizing agent for antiserum production. TEM showed rod shaped particles with 300 x 18 nm dimensions. The titer of produced antiserum was 1:1024 evaluated by microprecipitin test and indirect-ELISA. Coat protein gene was amplified by RT-PCR with expected size of (Approx. 500 bp). The PCR product was sequenced then the generated nucleotide sequence  was translated into 160 amino acids. The amino acid sequence of Five B-cell epitopes, of 14 amino acid residues each, were predicted. Identifying Bcell epitopes play an important role in vaccine design, immunodiagnostic tests, and antibody production. Therefore, computational tools for reliably predicting B-cell epitopes are studied.