Spectral and acid–base properties of 7-hydroxyflavone (7HF) in the ground and excited states were investigated with a purpose to enable reasonable application of this dye and its derivatives as fluorescent probes. Analysis of solvatochromic and solvatofluorochromic ability of 7HF in 20 solvents, investigations of 7HF spectral properties in the frozen solvents, spectrophotometric and spectrofluorimetric titrations in methanol–water (4:1 v/v) in the wide pH/H₀ range (from pH = 11.0 to H₀ = −4.5), analysis of the 3D fluorescence and time-resolved spectra, as well as quantum-chemical calculations were carried out. It has been found that 7HF can exist in three protolythic formsneutral, anion, and cationdepending on the environment acidity or basicity. In the excited state, in methanol–water solutions, there are four forms: neutral, cation and anion, which can be formed by direct excitation of the ground-state anion or by photodissociation of the neutral form depending on pH, and only one phototautomer, which appears in the H₀ range from 1.3 to −4.5. It has been shown that the mechanism of the phototautomer formation depends on medium acidity. The photoautomer can be formed by cation photodissociation as well as by photoanion protonation. Finally, it was concluded which of the 7HF protolytic forms can be used for fluorescent probing.