A two-photon excited fluorescent probe CMB-1 has been rationally developed for the detection and regeneration of formaldehyde based on a novel nucleophilic addition of a secondary amine to FA and subsequential alcoholysis reactivity mechanism. It enables a specific turn-on response towards formaldehyde and facilitates the monitoring of exogenous and endogenous formaldehyde in living cells via both one- and two-photon microscopy, with minimal influence on its native homeostasis and local concentration.