3-hydroxyflavone (3-HF) derivatives are very attractive fluorescence sensors due to their ability to respond to small changes in their microenvironment via a dramatic alteration of the relative intensities of their two well-separated emission bands. We developed fluorescence probes with locations at different depths and orientations of 3-HF moiety in the phospholipid bilayer, which determine their fluorescence behavior. While the spectral shifts of the probes correlate with their binding site polarity, the intensity ratio is a complex parameter that is also sensitive to the local hydration. We demonstrate that even the deeply located probes sense this hydration effect, which can be modulated by the charge of the lipid heads and is anisotropic with respect to the bilayer plane. Thus the two-band ratiometric fluorescence probes can provide multiparametric information on the properties of lipid membranes at different depths.