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The cellular ATP content level in agricultural products directly reflects cell viability, therefore it can be potentially used as an indicator of freshness and quality of agricultural products during storage. Spectral data of sample spinach leaves were obtained using a UV-Vis-NIR spectrophotometer UV-3600. Protoplast suspensions were prepared by following the conventional physical-chemical methods, and the ATP contents in protoplasts were determined by the firefly luciferase bioluminescence technology. Person's correlation analysis was performed to identify the key wavelengths. Models were developed for estimating the ATP contents in spinach protoplasts based on the two identified key wavelengths, i. e. the ultraviolet 298 nm and the near-infrared 730 nm wavelengths. Results showed that both of the two key wavelengths (298 and 730 nm) have a considerable promise in estimating the ATP content in spinach protoplasts (R2 = 0.802 9 and 0.901 respectively). The spectroscopy based estimation of cellular ATP content in vegetables proposed in this study provides a new approach to the accurate, rapid, and non-destructive evaluation of the freshness of vegetables.