The ELISA (Enzyme-Linked Immuno Sorbent Assay) chip to detect multiple allergic proteins simultaneously was developed successfully. This chip consists of multiple nitrocellulose membrane with 1mm diameter that immobilized several kinds of allergic proteins with 50ng amount only is fabricated using the multi-layer lithography. The experimental results demonstrated that 1) the specific allergen protein, and 2) the quantitative change of the antigen-antibody reaction depending on amount of antibody were able to be detected without contamination. The detection procedure that is only to inject antibodies in order into the chip is simple. The time for the whole detection procedure is short with 16 min. This ELISA chip that can specify the allergen protein that causes the patient's allergic disease is useful for the hyposensitization therapy.