An equimolar mixture of riboflavin-5′-phosphate sodium salt (RP) and adenine (AD) dissolved in a phosphate buffer (pH 4.0, 1.0% w/v) produces a red coloured transparent thixotropic hydrogel at 30 °C. The gelation of the RPAD system occurs in the pH range of 2–5. FTIR spectra and WAXS patterns indicate self-assembly via H-bonding between the >C=O group of RP and the amino/imino group of AD followed by supramolecular organization through a π-stacking process producing a fibrillar network structure. FESEM images clearly indicate that the nanofibres are produced from the intertwining of helical fibrils. The dynamic frequency sweep experiment of the supramolecular gel at a constant strain of 1% exhibits a wide linear viscoelastic region and a considerably higher G′ value (460 Pa) than that of G′′ (21 Pa) confirming the gel nature of the RPAD system. The hydrogel shows high stiffness (G′/G′′ = 3.3), a high yield stress (σ*) (79.5 Pa) and a moderate critical strain (γ = 17.5%). Time sweep experiments at both low (0.1%) and high strain (100%) indicate the thixotropic property of the gel. The RPAD hydrogel shows non-Newtonian viscosity in the shear rate region (0.1–158 s(−1)) and after that there is a sudden fall of viscosity. The gel melting point obtained by the falling ball method is 6° higher than that obtained by the DSC method probably due to the presence of the thixotropic property of the gel. The UV-vis spectra indicate a red shift of the π–π* transition band of RP in the RPAD xerogel. On excitation of the RPAD hydrogel at 373 nm it shows twelve times enhancement of emission intensity with a 7 nm red shift of the emission peak. This has been attributed to the enhancement of lifetime from 2.2 ns in RP to 3.4 ns in the RPAD hydrogel. With increase of temperature, the fluorescence intensity of the RPAD hydrogel at first increases till 40 °C, then decreases up to 55 °C and it again increases after 60 °C.