A detection kit for detecting an immunosuppressor in whole blood by high performance liquid chromatography-tandem mass spectrometry and a detection method thereof is provided. An internal standard solution is added with an antioxidant, vitamin E, and mixed with an internal standard diluent containing zinc sulfate heptahydrate, purified water and methanol for sample pretreatment, which not only exerts the function of the internal standard, but also synchronously achieves erythrocyte treatment, protein precipitation and target substance extraction. Various embodiments enable the immunosuppressor to be more stable in a solution matrix, thus promoting the detection accuracy and sensitivity. Various embodiments adopt isotopically-labeled sirolimus as an internal standard of everolimus to substitute isotopically-labeled everolimus, thus overcoming the interference of everolimus on isotopically-labeled everolimus and satisfying the detection requirements. Various embodiments detect four immunosuppressors simultaneously to reduce the cost of the internal standard, and has a lower detection cost, more accurate and stable detection results.