The invention discloses a CK-MB detection kit which comprises calibrator, cleaning fluid, a substrate solution, a pretreatment solution, an enzyme conjugate working solution and magnetic bead conjugate working solution, wherein the pretreatment solution contains imidazole, the enzyme conjugate working solution contains an enzyme marked CK-MB antibody, and the magnetic bead conjugate working solution contains CK-MB antibody marked magnetic beads. By means of the CK-MB detection kit, accurate measurement of CK-MB in a whole blood sample can be achieved, detection steps are simplified, and a detection efficiency is improved. The lowest detection limit of the kit is 1ng / ml, a linearity range is 1 to 300ng / ml, detection sensitivity is high, the linearity range is wide, and detection results areaccurate. The invention further discloses a use method of the CK-MB detection kit. Use steps of the use method are simple, CK-MB detection time is effectively shortened, and quick and flexible detection of the CK-MB is benefit to achieve.