Laser induced fluorescence (LIF) measurements have been performed in spray flames of diesel and rapeseed methyl ester (RME) using laser excitations of 266 nm, 355 nm, and 532 nm. Ultraviolet (UV) measurements in cold sprays of both fuels have also been carried out and are reported in this article. The LIF spectra obtained when exciting the cold spray of diesel with 266-nm and 355-nm laser excitations have been correlated to the presence of petrogenic polycyclic aromatic hydrocarbons (PAHs), which are known to be present in petroleum-derived fuels. Low fluorescence signals have also been detected for such excitation wavelengths in the RME spray. These signals have been related to the presence of trace species derived from the vegetable oil used to obtain the studied biodiesel. LIF signals obtained in flame conditions have been originally derived using a spectrally resolved multiple-excitation wavelength technique coupling laser-induced incandescence and fluorescence (LII/LIF). Such a technique is well adapted to isolate fluorescence emissions with a high signal/noise ratio in flame regions where soot and soot precursors coexist. A significant reduction of the LIF intensities has been observed for the three considered excitation wavelengths when using RME instead of diesel. The spectra obtained as a function of the height above the burner (HAB) with UV excitations are red-shifted in the diesel flame, while they exhibit the same spectral width in the RME one. This could be explained by the presence of other species in the RME flame, such as carbonyl compounds, that can interfere with the fluorescence of PAHs. LIF spectra at 532 nm present the same shape in both flames regardless of the HAB. Similar LIF spectra have also been obtained at specific positions in both flames with UV and visible excitations, which can be related to a similar distribution of PAHs. Thus, such a finding suggests that the heavy aromatic compounds that are related to the formation of nascent soot are similar despite the absence of petrogenic PAHs in RME.