The invention discloses a protein chip for detecting pathogen antibodies of blood and cerebrospinal fluid and its preparing method, and the protein chip includes substrate and peculiar-culture or shape protein or polypeptide antigen of array distributed pathogen and contrasted dot coating, where the substrate is a glass substrate; and the antigen and contrasted dot coating refers to 13 antigens with ten indexes including HSV-I, HSV-II, VZV, CMV, EBV, RV, JEV, MV, MT and LP, uniformly distributed on the glass substrate in a dot matrix form, and positive contrast, negative contrast and blank contrast. The protein chip of the invention can obtain multiple-index reacting result only by one reaction, judges infection of different pathogens and has the characters of quickness, high efficiency, accuracy and parallel diagnosis.