3D front-face fluorescence spectroscopy and principal component analysis (PCA) were used to differentiate between Extra Virgin Olive (EVOO) and Olive Oil (OO). The results showed that 3D front-face fluorescence could be an effective tool to characterize EVOO and OO. Fourteen samples of olive oils were acquired directly from producers and from retail markets and their 3D fluorescence spectra were measured and analyzed. The first principal component of the PCA model retained the most important variability as a combination of freshness due to the presence of antioxidant compounds and chlorophyll; and of oxidation stage due to the presence of oxidation products. Olive oil samples presented different spectral patterns providing different scores for EVOO and OO in the PCA model. EVOO samples selected according to the differences in their scores in the PCA were exposed to indirect light. The evolution of their emission spectra was monitored with a right-angle set-up showing differences in concordance with their scores. This technique, the 3D front-face, is useful for characterization of olive oil samples according to their fluorescent compounds. The combination with other techniques (1H HR-NMR, HPLC, NIR spectrometry, etc.) may also be useful in protocols devoted to decision making processes with regard to olive oil quality specifications.