The aim of this study was to test the usability of fluorescence spectroscopy to evaluate the stability of cold-pressed rapeseed oil during storage. Freshly-pressed rapeseed oil was stored in colorless and green glass bottles exposed to light, and in darkness for a period of 6 months. The quality deterioration of oils was evaluated on the basis of several chemical parameters (peroxide value, acid value, K232 and K270, polar compounds, tocopherols, carotenoids, pheophytins, oxygen concentration) and fluorescence. Parallel factor analysis (PARAFAC) of oil excitation-emission matrices revealed the presence of four fluorophores that showed different evolution throughout the storage period. The fluorescence study provided direct information about tocopherol and pheophytin degradation and revealed formation of a new fluorescent product. Principal component analysis (PCA) performed on analytical and fluorescence data showed that oxidation was more advanced in samples exposed to light due to the photo-induced processes; only a very minor effect of the bottle color was observed. Multiple linear regression (MLR) and partial least squares regression (PLSR) on the PARAFAC scores revealed a quantitative relationship between fluorescence and some of the chemical parameters.