The use of fluorescence spectra with 340 nm excitation was investigated for rapid evaluation of the quality attributes of beef musculus longissimus lumborum during chilled storage. The quality indices monitored were the Warner–Bratzler shear force, water-holding capacity (cooking loss and purge loss), pH, and color (L *, a *, and b *). The fluorescence characteristics of nicotinamide adenine dinucleotide (NADH) and porphyrin were highly correlated with these indices. A comprehensive and effective indicator was established by principal component analysis to reduce multicollinearity between each beef quality index. Quantitative models for prediction of the quality of beef were built using the partial least squares algorithm. The comprehensive indicator model provided satisfactory results with the R c 2 and R p 2 were 0.912 and 0.903, the RMSEC and RMSEP were 0.488 and 0.581. Therefore, this method could be used for a reliable assessment of beef quality during chilled storage. • 340 nm-excited fluorescence was used to evaluate beef quality during chill storage. • Fluorescence peaks of NADH and porphyrin were highly related to quality indices. • A comprehensive indicator for beef quality was established based on PCA. • Quantitative models combined with PLS gave satisfactory results. • A portable instrument could be developed with a specific wavelength. [ABSTRACT FROM AUTHOR]