Abstract: A protein chip, kit and preparation method thereof for detecting abnormal decarboxy prothrombin in serum, a substrate carrier of the protein chip is provided with a plurality of detection subareas; wherein each detection subarea is used for detecting a serum sample, and is internally provided with a detection-spot area and a control-spot area, and the detection-spot area has a detection spot formed by spraying a trace amount of a DCP-specific antibody, the control-spot area has a control spot formed by spraying a bovine serum albumin; all the detection spots within one of the detection-spot area have the same material concentration, to form each of the detection spots, a total volume of 3-5 nl of the DCP-specific antibody with a concentration of 3-5 mg/mL is used; each of the detection spots is formed by a non-contact spotter, performing 6-10 spot sprays and spraying 300-500 pL in each spray.