Although fish and its related products are good sources of protein and unsaturated fatty acids, like omega-3 in the human diet, their shelf-life is limited by biochemical and microbial changes. In this study, a front-face fluorescence spectroscopy technique was used to acquire Excitation-emission matrices (EEM) to monitor Japanese dace (Tribolodon hakonensis) fish freshness degradation during storage. EEM of Japanese dace fish parts (intact eyeball and surface-containing scales), excitation from 220 to 585 nm and emissions from 250 to 600 nm, were measured at different times during storage. To simplify the acquired complex spectra datasets from each fish part, the variables were reduced to those that were only significant/important (those with higher positive or negative correlation) for K value prediction, and as an index of freshness. Partial least square regression (PLSR) results demonstrated that combining the fluorescence EEM of the eyeball and surface-containing scales the best monitoring of fish freshness; excitation at 280 and 350 nm for both the eyeball and surface-containing scales, with 2.84 and 0.96 as RMSE and R2, respectively. These findings demonstrate that multiple excitation fluorescence approaches can be convenient for the freshness evaluation of fish.