The invention discloses diluent of carboxylated magnetic particles and a preparation method of the diluent. The diluent is prepared from, by weight, 39-55 parts of N-(2- ethoxyl)piperazine-N'-2-ethanesulfonic acid, 28-38 parts of dipotassium phosphate, 9-18 parts of ethylene diamine tetraacetic acid, 11-29 parts of zinc chloride, 7-12 parts of magnesium chloride, 6-13 parts of heparin sodium, 15-22 parts of saccharose, 0.1-0.3 part of hydrochloric acid, 4-12 parts of glucose, 1-3 parts of bovine serum albumin, 0.2-0.6 part of mouse serums, 0.3-0.7 part of glycerin, 0.2-0.4 part of Proclin 300,0.4-0.8 part of Tween-20 and 100-300 parts of deionized water. According to the diluent of the carboxylated magnetic particles and the preparation method of the diluent, through synergistic effect ofall the components, the diluent of the carboxylated magnetic particles is prepared and used for diluting carboxylated magnetic particle mother liquor into a working solution, the working solution canbe stored for a long time, and the specificity, stability, repeatability and other performance of a reagent are not influenced.