A reagent for extracting immunosuppressant drugs from a whole blood sample for immunoassay includes protein denaturant, proteolytic enzyme, surfactant and pH buffer. A method and an immunoassay kit for detection of the immunosuppressant concentration in a whole blood sample uses the extraction reagent. The extraction reagent doesn't need the use of organic solvent as that in the traditional extraction methods, therefore the adverse effects of the organic solvent on the antibody activity in a detection system and the other relative defects associated to its use are obviated. The drug extraction process doesn't need centrifugation, as the processed sample can be directly applied for immunoassay. The operation for drug extraction is simple, and the detection result based on this extraction method is accurate.