Highlights • 3-D fluorescence spectroscopy was used to monitor beef deteriorative changes. • Correlation between beef quality and fluorescence EEM peaks were investigated. • Rapid and non-destructive approach for evaluation of beef freshness. • The method is significant to protect consumers and ensure food security. Abstract We investigated three-dimensional (3-D) fluorescence spectroscopy for its potential to evaluate beef quality deteriorative changes and freshness. The fluorescence characteristics of heme, conjugated Schiff base and amino acids, could be indicators of internal biochemical reactions associated with beef deterioration, including color changes, lipid oxidation, and protein degradation, as well as a measure of freshness decline. To classify beef quality in terms of color (sensory index) and pH (chemical index), cluster analysis method (CA) was used. Three classes were identified: "fresh", "acceptable", "spoiled". We then developed a qualitative model to classify stored beef into these three classes using 3-D front-face excitation-emission matrices (EEMs) of fat tissue, combined with a parallel factor analysis (PARAFAC) algorithm. The resulting model had calibration and validation accuracies of 95.56% and 93.33%, respectively. These results demonstrate the potential of fluorescence spectroscopy to accurately and non-destructively monitor beef quality decline. [ABSTRACT FROM AUTHOR]