Various aggregation-induced emission luminogens (AIEgens) have been developed and applied in different areas in recent years. However, AIEgens generally can aggregate and emit strong fluorescence in aqueous solution even containing DNA and other biomacromolecules because of poor water solubility, restricting their application in biosensing and bioimaging in aqueous solution. Moreover, the great majority of AIEgens commonly suffer from complex organic synthesis, environmental damage, and biological toxicity. In this work, jatrorrhizine (Jat), an isoquinoline alkaloid from Chinese herbs, was found to be a natural water-soluble AIEgen that has not been previously reported. Jat’s photometric characteristics and single-crystal structure demonstrated that the restriction of intramolecular motion and twisted intramolecular charge transfer were responsible for its AIE phenomenon. Due to the good water solubility and AIE character of Jat, it did not emit fluorescence in the aqueous solution containing DNA and polymers until the formation of the DNA hydrogel. Therefore, a DNA hydrogel fluorescence biosensor was designed by using the target (miRNA) as a catalyst to trigger the entropy-driven circuit of DNA, realizing the ultrasensitive and label-free detection of miRNA with an ultralow limit of detection (0.049 fM, S/N = 3). This biosensing strategy also has excellent stability and acceptable reliability for real sample assay. The results not only indicated the excellent sensing performance of Jat as AIE probes in aqueous solution but also demonstrated the promising application potential of water-soluble natural AIEgens. © 2023 American Chemical Society