Bioactive molecules are highly worthwhile to recognize and explore the latent pathogenic mechanism. Conventional methods for bioactive molecule detection, including mass spectrometry and fluorescent probe imaging, are limited due to the complex processing and signal interference. Here, we designed enzyme-reaction-assisted programmable transcriptional switches for the detection of bioactive molecules. The approach is based on the use of programmable enzyme site-specific cleavage-assisted DNA triplex-based conformational switches that, upon responding to bioactive molecules, can trigger the transcription of fluorescent light-up aptamers. Thanks to the programmable nature of the sensing platform, the method can be adapted to different bioactive molecules, and we demonstrated the enzyme-small molecule catalytic reaction combination of myeloperoxidase (MPO)-hydrogen peroxide (H2O2) as a model that transcriptional switches was capable of detecting H2O2 and possessed the specificity and anti-interference ability in vitro. Furthermore, we successfully applied the switches into cells to observe the detection feasibility in vivo, and dynamically monitored changes of H2O2 in cellular oxidative stress levels. Therefore, we attempt to amalgamate the advantages of enzyme reaction with the pluripotency of programmable transcriptional switches, which can take both fields a step further, which may promote the research of biostimuli and the construction of DNA molecular devices.