Nitrite (NO2-) has been widely used in the food and beverages manufacturing processes. It has been identified as a significant threat chemical to human health and heavily assayed in the field of food safety and water quality control. Using Griess-reaction to produce dizaonium salt or initiate further cyclization reaction for signalling are the mainstream strategies for NO2- sensing. However, conventional amino recognition reaction sites often suffer from extensive incubation time and strong acidic medium. Herein, we developed a diaminosubstituted cyclohexadiene fluorescent probe (DAE) by “multicomponent one pot” synthesis. The as-prepared probe DAE is endowed with two amines activated by carboxylic acid ester, making it easy to react with NO2-. DAE exhibited sensitive and specific response to NO2- in a weak acid medium (acetonitrile-PBS system, pH 6.0) due to the activation of adjacent carboxylic ester groups. A DAE-based method for the detection of NO2- was established, offering a LOD of 7.75 nM. Preliminary mechanism investigation reveals that this specific recognition reaction resulted from the NO2--mediated diazotization and decarboxylation ester of DAE. Moreover, the DAE-loaded paper device was fabricated and employed to monitor NO2-, featuring simplicity and high efficiency. DAE was also successfully employed for fluorescence imaging in live cells, displaying good biocompatibility and excellent imaging ability. Overall, the developed probe DAE has addressed the difficulties of long incubation time and strong acid condition associated with the Griess-based probes, representing a prospective fluorescent probe for NO2- sensing.. https://www.jscimedcentral.com/public/assets/images/uploads/image-1760676513-1.PNG Highlights 1. A diaminosubstituted cyclohexadiene fluorescent probe (DAE) was developed. 2. DAE contains activated dual recognition sites for the reaction with NO2-. 3. DAE shows highly sensitivity and selectivity toward NO2- in a weak acid medium. 4. DAE was also employed for fluorescence imaging in live cells.