Formaldehyde is widely used in daily life and can be generated endogenously through diverse biological processes. However, formaldehyde may cause severe threat to the health of humanity. In this work, we designed a novel nanoprobe NaP@Pβ-CD through the host-guest self-assembly between poly β-cyclodextrins (Pβ-CD) and naphthalimide (NaP). The NaP is enclosed in the Pβ-CD cavity, forming hydrophilic nanoparticles. Formaldehyde can trigger “on” fluorescence response of NaP fluorophore and the detection can be carried out in aqueous solution. This probe has low detection limit (16.8 nM), a fast response to FA (5 min), high selectivity, good biocompatibility and excellent stability. More importantly, it shows excellent sensing capability totally in water without requiring any organic solvent. Therefore, the NaP@Pβ-CD could be successfully applied to formaldehyde detection in food, living cells, and organisms in vivo.