We report on time-dependent Stokes shift measurements of single molecules. Excitation and emission spectroscopy were applied to study the temporal Stokes shift evolution of single perylene diimide molecules embedded in a polymer matrix on the time scale of seconds. The Stokes shift varied between individual molecules as well as for single molecules undergoing different conformations and geometries. From the distribution and temporal evolution of Stokes shifts, we unravel the interplay of nanoenvironment and molecular conformation. We found that Stokes shift fluctuations are related to simultaneous and unidirectional shifts of both emission and excitation spectra.