Substitution of frozen-thawed food products for fresh ones is a significant authenticity issue being extensively investigated over the past few years by various conventional methods, but little success has been achieved. Fluorescence spectroscopy is a sensitive and selective spectroscopic technique that has been widely applied recently to deal with various food quality and authenticity issues. The technique is based on the excitation of certain photosensitive components (known as fluorophores) to fluoresce in the UV and visible spectral ranges. Fluorescence spectroscopy can be performed to obtain simple classical two-dimensional fluorescence spectra (excitation/emission), synchronous or three-dimensional excitation–emission matrices (excitation/emission/fluorescence signal). The technique can be used in front-face or right-angle configurations and can be even combined with hyperspectral imaging, requiring the use of multivariate data analysis to extract useful information. In this review, we summarize the recent progress in applications of fluorescence spectroscopy to differentiate truly fresh foods from frozen-thawed products. The basics of the technique will be briefly presented and some relevant examples, focusing especially on fish and meat products, will be given. It is believed that interdisciplinary collaboration between researchers working with data analysis and spectroscopy, as well as industry and regulatory authorities would help to overcome the current shortcomings, holding the great promise of fluorescence spectroscopy for fighting food fraud in the food industry.