Four water‐soluble 2‐hydroxy‐Nile Blue derivatives, 1 a, 1 b, 2 a, and 2 b, were prepared by condensation reactions performed under relatively mild conditions (90 °C, N,N‐dimethylformamide with no added acid). These fluorescent probes had more favorable fluorescence characteristics than two known water‐soluble Nile Blue derivatives. Specifically, they were superior to the known dyes with respect to their quantum yields in aqueous media and the sharpness of their fluorescence emissions. Concentration‐dependant UV absorption and fluorescence emission studies indicated that the dyes did not aggregate in aqueous solution at concentrations of less than 1–4 μM. The new water‐soluble materials 1 a, 1 b, 2 a, and 2 b emit in a desirable region of the fluorescence spectrum (λ=670–675 nm). Overall they are potentially interesting for labeling biomolecules in aqueous environments.