The invention discloses a calprotectin chemiluminiscence immunoassay kit and a preparation method thereof, belongs to the field of in-vitro detection, and solves the problems of low sensitivity, highcost, poor accuracy, long operation time, low automation degree and incapability of quantification of an existing kit. The chemiluminescence immunoassay kit comprises streptavidin magnetic particles,a chemiluminescence marker labeled calprotectin antibody, a coupling marker labeled calprotectin antibody, a chemiluminescence substrate solution and a calprotectin calibrator, wherein the concentration of the magnetic beads is 0.02-1%, and the particle size is 1.0-3.0 microns, the molar ratio of the calprotectin antibody to the chemiluminiscence marker is 1: (3-20), the molar ratio of the calprotectin antibody to the coupling marker is 1: (5-20), the chemiluminescence marker is acridinium ester, luminol, isoluminol or terpyridyl ruthenium. And the coupling marker is biotin. The method is highin sensitivity, high in accuracy, strong in specificity, high in automatic measurement degree, low in cost, small in error, short in measurement time and wide in application range.