The invention relates to the technical field of urine special protein detection, in particular to a urine special protein composite quality control substance and a preparation method thereof, the quality control substance comprises a low-value positive quality control substance and a high-value positive quality control substance, and the low-value positive quality control substance and the high-value positive quality control substance respectively comprise a buffer agent, urea, salts, a preservative, creatinine, PH, a protein protective agent, a stabilizer, human albumin, transferrin, retinolconjugated protein, alpha1-microglobulin, beta2-microglobulin, immune globulin G, neutrophil gelatinase associated lipocalin. The urine special protein composite quality control substance and the preparation method thereof provided by the invention have the advantages of simple operation, instant use after decapping, good stability, low production cost and easiness to achieve the quality control of a urine special protein detection system.