The invention discloses a method for preparing a temperature stimuli responsive intelligent chromatographic column, which comprises the following steps of: A, uniformly mixing N-Isopropyl acrylamide, N,N'-methylene-bis-acrylamide, polyethylene glycol-20,000, 10 percent w/v ammonium persulfate and deionized water by using ultrasonic waves; B, deoxidizing reaction products in the step A, adding 10 percent v/v N,N,N',N'-tetramethyl ethylene diamine, and uniformly mixing fully; and C, pouring reaction products in the step B into a stainless steel chromatographic column tube, performing reaction at the temperature of between -18 and 60 DEG C for 10 to 100 hours, arranging chromatographic column heads at two ends of the column tube, connecting the column tube to a high pressure pump, and washing the chromatographic column body by using ultrapure water as a mobile phase. The method has the advantage of simple process; the prepared monolithic column has good pore structure, high mechanical strength and high column efficiency; during use, the water is taken as the mobile phase, so the waste and pollution caused by using a large quantity of organic solvent can be avoided; and in the separation process, the action force between reagents and the monolithic column can be controlled only by changing temperature, so the separation is simple and feasible, and the separation effect is good.