© 2017 by the authors. Licensee MDPI, Basel, Switzerland. Red sea bream (Pagrus major) scale fluorescence characteristics were identified as a potential rapid and non-destructive means for assessing the fish’s freshness. To investigate this, live red sea breams were purchased, slaughtered, and prior to measurement, stored at 22 ± 2◦C for 27 h. During subsequent storage, the K value of the dorsal meat—as a standard freshness indicator—along with front-face fluorescence spectra of representative dorsal scales, were measured simultaneously at 3 h intervals. Two major fluorescent peaks, A and B, were identified with excitation and emission wavelength pairs of 280/310 nm and 340/420 nm, which were mainly contributed to by tyrosine and collagen, respectively. Subsequent analysis showed that the fluorescence intensity ratio of peak B to A (IB/IA) increased linearly during storage (R2 = 0.95) and is proposed as a potential non-destructive index of fish freshness. Thus, our results suggest that the fluorescence characteristics of fish scales can be used to assess fish carcass freshness during storage.