Conventionally, biochemical analysis is used to estimate the freshness of fish products, but this method is destructive, technically demanding and time consuming. In this study, the fluorescence properties of raw fisheyes of Japanese dace (Tribolodon hakonensis) fish were investigated using spectrofluorometer during 4 degrees C storage. Subsequently, according to the results of the spectrofluommeter, fluorescence imaging system was established with 365 nm UV LED to capture the fluorescence images using a common color camera. The color components from fluorescence images of pupil and iris were extracted to establish a prediction model for a 'standard freshness index' of K value which was acquired by using biochemical analysis using partial least square regression method. From the results, RMSECV of 3.5% and R-2 of 0.92 was realized. This study demonstrated that the UV-fluorescence imaging technique has a potential to estimate the fish freshness non-destructively during storage.