The invention provides a kit for accurately measuring the concentration of four immunosuppressant drugs in human whole blood. The kit comprises the calibrators of tacrolimus, sirolimus, everolimus andcyclosporine A, the internal standards of the tacrolimus-13Cd2, the sirolimus-d3, the everolimus-d4 and the cyclosporine A, the quality control serums of the tacrolimus, the sirolimus, the everolimusand the cyclosporine A, sample treatment liquid, a 96-well plate assembly, and a mobile phase. According to the kit and detecting method for accurately measuring the concentration of four immunosuppressant drugs in human whole blood, a clean treatment liquid can be obtained through a simple 96-well plate protein precipitation pretreatment method without complex purification steps, and the required sample amount is small and can be used together with a liquid chromatography-tandem mass spectrometry; and four different immunosuppressant drugs can be detected at the same time, that is, the single-sample multi-index synchronous detection can be realized, the detection specificity is good, the sensitivity is high, the whole detection time is short, the throughput is high, and the cost is low.