Gene therapeutic approaches are needed that can simultaneously induce the well-controlled expression of therapeutic genes and suppress the expression of disease-causing genes for maximization of their efficacy. To address this challenge, we designed an allosteric ribozyme that comprises a Tetrahymena group I-based trans-splicing ribozyme as an active domain for RNA replacement, a small molecule-specific RNA aptamer as a sensor domain, and a communication module as an active transfer domain. The effectiveness of this approach was assessed by constructing various ribozymes in combination with a theophylline-binding aptamer to identify an allosteric ribozyme, which is controlled by theophylline both in vitro and in cells. Moreover, we constructed adenoviral vectors encoding the ribozymes and validated allosteric regulation of trans-gene expression via theophylline-dependent RNA replacement in target RNA-expressing cells. Results demonstrate that an allosteric trans-splicing ribozyme is an applicable RNA-based framework for engineering external ligand-controlled gene expression regulatory systems that exhibit adjustable regulation, design modularity, and target specificity.