The invention relates to a preparation method of an alpha-oxoglutarate fluorescent / ultraviolet molecular probe and application of the alpha-oxoglutarate fluorescent / ultraviolet molecular probe to biological samples. The preparation method includes the steps that NBD-Cl is dissolved in chloroform, the dissolution concentration is 0002-0.012 g / ml, then a hydrazine hydrate-methanol solution with the volume concentration being 0.2-1.2% is added in and uniformly mixed, brown precipitate is obtained through stirring at room temperature and filtered, a filter cake is washed through ethyl acetate and dried, and the brown product alpha-oxoglutarate fluorescent / ultraviolet molecular probe is obtained. The alpha-oxoglutarate fluorescent / ultraviolet molecular probe is applicable to qualitatively and quantitatively analyzing alpha-oxoglutarate in the biological samples, and detection is sensitive, accurate and fast. The biological samples mainly comprise serum, living cells, muscle tissue and the like. The alpha-oxoglutarate fluorescent / ultraviolet molecular probe can be applied to analytical chemistry, life organic analysis, disease pre-diagnosis, medical clinical inspection and other related fields. The alpha-oxoglutarate fluorescent / ultraviolet molecular probe has the advantages of being good in response performance and high in data accuracy, repeatability and precision, equipment is convenient, fast and easy to operate, operability is high, and the alpha-oxoglutarate fluorescent / ultraviolet molecular probe is particularly suitable for large-batch sample combined screening and other large-data research.