Mixed virus infection has increasingly become a problem in the production of Solanaceae crops in recent years; therefore, a fast and accurate detection method is needed. In this study, a novel triplex immunostrip assay was developed for the simultaneous detection of tobacco mosaic virus (TMV), tobacco vein banding mosaic virus (TVBMV), and potato virus Y (PVY). The limits of detection of this novel immunostrip reached 200 ppb (ng/ml), 1 ppm (µg/ml), and 2 ppm for TMV, PVY, and TVBMV particles, respectively. Importantly, no cross-reactivity was observed among TMV, TVBMV, and PVY or to a nontarget virus. When the assay was applied to suspected virus-infected tobacco, tomato, and potato samples collected from fields in Southwest China, samples of single or mixed virus infection were successfully identified. In conclusion, the triplex immunostrip assay provides a fast and easy to use on-site detection method for field epidemiological studies of TMV, TVBMV, and PVY, and for managing diseases that are caused by them.