Plant systemic resistance induced by natural product is an alternative technique of disease management. Riboflavin (vitamin B2) usually used as abiotic elicitor to improve the plant immunity against different pathogens. This work aimed to explore the efficiency of three riboflavin concentrations (0.5, 2.5, and 5 mM) to enhance resistance toward Tobacco Mosaic Virus (TMV) in tomato plants. Our results showed that exogenous application of 2.5mM riboflavin, 5 days before virus challenge was the most effective concentration, which provided a reduction in disease incidence and disease severity by 80% and 75%, respectively. Furthermore, obtained results were confirmed by using DAS-ELISA test, which showed that concentration 2.5 mM of riboflavin decreased the virus concentrations by 46.4% in treated- inoculated plants. It was remarked that exogenous application of 2.5 mM riboflavin showed a positive effect on some plant growth parameters either in presence or absence of TMV challenge. The plant height and number of leaves per plant were significantly improved in treated- inoculated plants about 30% and in compared to the infected control 78%. In time course investigation, 2.5mM riboflavin treatment reduced the virus symptoms particularly at 9th day, where after the symptoms become evident. In addition, expression of phenylalanine ammonia-lyase (PAL) and pathogenesis-related protein (PR10), which are the markers of systemic acquired resistance (SAR), were rapidly increased in inoculated-treated tomato from 1-3 days after treatment. Moreover, the results of the biochemical changes analysis revealed that, the levels of the defense markers including peroxidase (PO), and polyphenol oxidase (PPO) were increased four days after of treatment and reached maximum levels at 8 days in the treated- inoculated plants. In conclusion, it could be suggesting that riboflavin exerted a great influence on TMV disease, as indicated by reduction of disease symptoms as well as enhancement of biochemical changes in plant defense against infection with TMV.