Abstract A novel bioluminescence (BL) assay of pyruvate phosphate dikinase (PPDK) has been developed using the firefly luciferin–luciferase reaction. PPDK catalyzes the formation of ATP from AMP, PPi (diphosphate) and phosphoenolpyruvate (PEP). The BL assay for PPDK was highly sensitive with a low background because it was not affected by adenylate kinase, which generates ATP from 2 mol of ADP and is present in various microorganisms. The proposed assay was performed as follows: 10 μl of standard PPDK solution was added to a microtiter plate, followed by addition of 100 μl of bioluminescent solution (containing AMP, PPi, PEP, Mg 2+, luciferin and luciferase). The BL intensity was integrated for 5 s after incubation for 15 min. The range of log–log calibration linearity and 3S.D. detection limit of PPDK ranged from 3.5×10 −20 to 4.5×10 −16 and 1.36×10 −20 mol per assay, respectively. The within-assay precision for PPDK was 1.2–2.8% ( n=8). The BL intensity was stable for at least 120 min at 37°C. The proposed assay to was applied to a BL enzyme immunoassay (EIA) for α-fetoprotein (AFP) and insulin using PPDK as the enzyme label. The detection limits for AFP and insulin were 2.79×10 −18 and 9.26×10 −17 mol per assay, respectively. Serum samples could accurately be analyzed by the BL-EIA. A high degree of correlation was observed between the values obtained using the proposed BL-EIA and conventional methods.