An important challenge for reaction-based fluorescent probes is that they generally require analyte consumption for fluorescence signal generation, thus creating potential perturbation of native analyte homeostasis or change of local concentrations. Herein, we reported two formaldehyde (FA) regeneration fluorescent probes, NAP-FAP-1 and NAP-FAP-2. An unprecedented regiospecific FA-induced intramolecularity strategy is implemented in the probe design, which adopts 3-(benzylamino)-succinimide as the FA-selective reaction group. The probes are able to capture the analyte molecule, induce regiospecific imide bond cleavage, and then release the captured FA molecule with simultaneous fluorescence turn-on response via a unique dual PeT/ICT quenching mechanism. The probes have shown potentials in detection, comparison, and imaging of FA levels intracellularly and inside lysosomes. These features make them useful for the study of FA homeostasis and functions in biological systems with minimal perturbation.