The invention discloses a preparation method of quality control serum for quality control of centrifugal microfluidic chips. The method comprises the following steps: adding 0.6 to 1.0 percent of cholesteryl sodium sulfate into bovine plasma, and adding 0.02 to 0.06 percent of monopotassium phosphate; sequentially adding 0.04 to 0. 08 percent of ammonium ferric sulfate dodecahydrate, 0.5 to 0.9 percent of calcium chloride, 0.2 to 0.6 percent of bitter salt, 0.6 to 1 percent of urea, 9 to 13 percent of sodium chloride, 0.01 to 0.03 percent of zinc vitriol, 0.01 to 0.03 percent of chalcanthite,0.06 to 0.2 percent of glycocholic acid, 2 to 4 percent of glucose, 0.03 to 0.06 percent of creatinine, 0.08 to 0.2 percent of uric acid, and 0.07 to 0.11 percent of triolein into water; uniformly mixing obtained solution; adding glycol, saccharose and triton X-100, and uniformly mixing; sequentially adding albumin bovine serum, sodium azide, alanine aminotransferase, aspartic transaminase, alkaline phosphatase, lipase and creatine kinase, uniformly mixing, and performing freeze drying. The method has the advantages that the source of the raw materials is sufficient, the raw materials are easyto get, possible matrix effect is furthest avoided, and precipitation of the raw materials is prevented.