Detection of gene expression in plants is critical for understanding the molecular basis of complex plant biosystems and plant responses to environmental stresses. Here, we report the development of a split ribozyme-based biosensor that enablesin vivovisualization of gene expression in plants. We demonstrated the utility of this biosensor in transient expression experiments (i.e., leaf infiltration inNicotiana benthamiana) to detect RNAs derived from transgenes and tobacco rattle virus, respectively. Furthermore, we successfully engineered a split ribozyme-based biosensor inArabidopsis thalianaforin vivovisualization of endogenous gene expression at the cellular level. In addition, we developed a platform for easy incorporation of different reporters into the RNA biosensor.