Formaldehyde (FA, HCHO) is a highly reactive carbonyl species, which is very harmful to humans and the environment as a tissue fixative and preservative. Therefore, developing some highly sensitive, selective, and rapid detection methods is significant for human health in food safety and environmental protection. Herein, a two-photon (TP) ratiometric sensor, CmNp-CHO, has been constructed by conjugating a TP donor (Π-push-pull-structure) with a FA off-on acceptor (functioned with hydrazide moiety) via a nonconjugated linker through the fluorescence resonance energy transfer mechanism. Such a scaffold affords CmNp-CHO a reliable and specific probe for detecting FA with two well-resolved emission peaks separated by 124 nm. Also, it responds to FA rapidly with high selectivity and sensitivity during 1.0 min and a large ratio enhancement at I550/I426 with addition of 0-20μM FA, exhibiting ∼4-fold ratio increase and a fairly low LOD of 8.3 ± 0.3 nM. Moreover, CmNp-CHO has been successfully employed for detecting FA in live cells, onion tissues, and zebrafish, exhibiting that CmNp-CHO can serve as a useful tool for investigating FA in real food application and offering strong theoretical support and technical means for investigation of physiological and pathological functions of FA.