The invention discloses a 25,27-di(3-methyl-thio-ethoxy thiadiazole) calix[4]arene stationary phase which is low in cost, high in content of compounded calixarene bonded stationary phase bonded amount, and stable in stability bonding layer, and is prepared by the following steps of: under the action of phase transfer catalyst ammonium tetrabutyl bromide, carrying out nitrogen protection to gamma-glycidol ether bonded silica gel and calixarene derivative sodium phenolate, and preparing the object calixarene stationary phase at 80 DEG C. The calixarene stationary phase compounded by the method in the invention is high in bonded amount, stable in boned layer, simple and easy in compounding method, low in cost, free of pollution in the compounding process and the like. The stationary phase not only has traditional ODS (Octadecylsilyl) reversion phase chromatography performance, and has containing complexing action, pi-pi action, hydrogen bonding action and dipole-dipole action because of many action sites; in a certain degree, the stationary phase in the invention has special separation performance comparing with the traditional ODS column.