The invention relates to a preparation method of an inorganic-organic hybrid monolithic column. The preparation method comprises the following steps: A. adjusting the pH value of Fe<2+> compound containing aqueous solution, adding with nano-diamond, adding with H2O2 and reacting to obtain hydroxyl nano diamond; B. mixing the hydroxyl nano diamond with KH570, dissolving into ethyl alcohol, carrying out ultrasonic treatment, reacting, centrifuging, washing, and drying to obtain the coupled hydroxylized nano diamond; C. mixing the coupled hydroxylized nano diamond, dipentaerythritolhexaacrylate, a cross-linking agent, an initiator and a pore-foaming agent, injecting the mixture into a stainless steel column, and reacting for 1-1.5h; and D. connecting the stainless steel column with a high-pressure infusion pump, flushing, and drying to obtain the inorganic-organic hybrid monolithic column. The preparation method has simple process, the reaction condition can be realized easily, the operability is good, the prepared monolithic column has uniform framework and pore diameter, has good separation effect and can be used for separating complex samples under a wide-temperature-range condition, and the application field of the monolithic column can be expanded greatly.