The invention provides a method for enzyme activity analysis and detection of pyruvate dehydrogenase complex (PDH complex, PDHC), and the invention also provides a reagent for determining the enzyme activity of PDHC. The method and the agent can be used for analysis and determination of the enzyme activity of PDHC in samples, and thus can be used for analysis, examination and diagnosis of clinical diseases. The principle of the invention is that: PDHC can orderly perform catalytic decarboxylation of pyruvic acid to generate products of CO2, acetyl coenzyme A, and NADH. Therefore, detection of the generation of the final product NADH means the detection of the enzyme activity of PDHC. NADH can emit fluorescence at a specific excitation wavelength, and the reaction rate is in direct proportion to the product fluorescence intensity, so the enzyme activity of PDHC in a sample can be calculated by detecting the fluorescence value change. The detection method of the enzyme activity of PDHC provided in the invention can realize rapid, specific, and sensitive determination of the enzyme activity of PDHC in a sample. Based on the method, the reagent for determining the enzyme activity of PDHC provided by the invention has the characteristics of convenience, rapidness, and high sensitivity, and facilitates popularization and application.