Intrinsic fluorescence from L-tryptophan (L-Trp) is routinely used to obtain insight into the structural features and dynamics of proteins and enzymes. In contrast to aqueous enzymology, different parameters that control and influence the behavior of proteins and enzymes in nonaqueous media depend heavily on the solvent. Detailed analysis of the intrinsic fluorescence from L-Trp dissolved in two deep eutectic solvents (DESs), reline and glyceline, prepared by mixing salt choline chloride with H-bond donors urea and glycerol, respectively, in a 1:2 molar ratio within 298.15-358.15 K temperature range, is presented. Fluorescence emission maxima of L-Trp dissolved in DESs show bathochromic shift with increasing temperature. In comparison to water and several organic solvents, the fluorescence quantum yields of L-Trp in both DESs are significantly higher. While the rates of nonradiative decay in the two DESs are comparable and increase with increasing temperature, radiative decay rates are independent of temperature and are higher in glyceline than in reline, resulting in a higher fluorescence quantum yield of L-Trp in glyceline. Excited-state emission intensity decays of L-Trp fit best to a double exponential model irrespective of the identity of the DES and temperature. Average lifetime decreases with increasing temperature due to increased thermal deactivation; however, this decrease is much slower in DESs as compared to that in water. Both steady-state fluorescence anisotropy and rotational reorientation times for L-Trp are governed by the inherent complexity of the DESs as solubilizing milieu resulting in noncompliance to simple hydrodynamic treatment. Fluorescence quenching of L-Trp by acrylamide in reline is purely dynamic in nature. This is in contrast to the aqueous media where the decrease in fluorescence is a combined result of both dynamic and static quenching. The quenching within reline is fairly efficient considering the high viscosity of the medium. Significantly lower activation energy of the bimolecular quenching process as compared to the activation energy of the viscous flow indicates facilitation of the electron/charge transfer quenching of L-Trp by acrylamide within the ionic environment offered by reline. The effect of high viscosity is partly overcome by the strongly ionic environment of reline during the electron/charge transfer between L-Trp and acrylamide. The results highlight the structural complexity of these DESs especially within the cybotactic region of the probe, which is absent in common molecular solvents of similar high viscosity.