A sensitive bioluminescent enzyme immunoassay (BLEIA) has been developed using acetate kinase(AK) as a new label enzyme. The bioluminescent assay of AK was done by firefly luciferase for measuring ATP produced by enzymatic reaction of AK using acetylphosphate and ADP as substrate. The detection limit of AK was 60 zmol/assay(1.2 amol/ml) and the coefficient of variation (CV, n=5) for each point, ranged from 2.1 to 8.3%. The proposed bioluminescent assay of AK was very simple, rapid and highly sensitive. The AK assay developed here, was applied to BLEIA of 17 α-hydroxyprogesterone, rabbit IgG and human thyroid stimulating hormone(TSH) as model compound for competitive and immunometric immunoassay.