Abstract: Objective: To establish an enzyme-linked immunosorbent assay (ELISA) for detection of the antibody of penicillin in human serum. Methods: The ELISA to detect the penicillin antibody was established by adding sera into the polystyrene micro-titer plates coated with penicillin-conjugated rabbit serum albumin and HRP-conjugated goat anti-human IgG. The Penicillin antibody in some clinical samples was detected with this method. Results: The within and between assay CV were 6.8% and 8.4% respectively. The rabbit anti-penicillin antibody, which was obtained from penicillin-conjugated rabbit serum albumin, could control the HRP-anti human IgG reaction. The positive rate of penicillin antibody in patients with different diseases was significantly different. Conclusions: ELISA is more sensitive and specific in clinically detecting and antibody of penicillin.