MicroRNAs (miRNAs) as novel biological targets are hardly applied in diagnostic and treatment of diseases, as they are difficult to be accurately detected and regulated. Here, we demonstrated a modular DNA intelligent machine named three-dimensional tweezers (TD-tweezers) to image and regulate miRNAs in living cells simultaneously. Fluorophore or miRNA inhibitors are introduced as detecting or regulating parts to construct different types of TD-tweezers, and the conformational state of TD-tweezers is controlled by the target miRNAs. The TD-tweezers exhibit excellent sensitivity, specificity, stability, and biocompatibility in vitro and in vivo, and their function of regulating miRNAs was confirmed by the up-regulated expression of downstream genes and proteins. Moreover, the TD-tweezers have been tested in whole blood, preliminarily verifying their clinical application potential. This design provides a multifunctional platform that can achieve efficient detection and regulation of targets within living cells and promote the development of DNA intelligent machines.