The invention relates to a doxofylline injection-related substance determination method. According to the method, a high-efficiency liquid chromatography is used for determination. The invention provides a method capable of effectively controlling the doxofylline injection-related substances by using the high-efficiency liquid chromatography, which has the beneficial effects being good in durability, simple and rapid in operation, high in system suitability. According to the method, at least three reference substances containing theophylline, theophylline ethanol and theophylline acetaldehyde are used for the determination of the related substances. At the same time, the invention provides impurity reference substances such as impurity A, impurity B, impurity C, impurity D, impurity E, impurity G, impurity caffeine, methylxanthine, impurity C', impurity D', impurity E', impurity 2, theophylline acetic acid and the like for the determination of the doxofylline injection-related substances, so that the related substances caused by different synthetic routes, and preparation processes and storage processes of preparations. The invention also provides a synthesis method for separating and synthesizing the reference substances containing impurity A, impurity A'', impurity B, impurity C, impurity D, impurity E, impurity G and the like for the determination of the doxofylline injection-related substances.