A new type of fluorescence assay for the determination of peroxidase (POx) activity is presented. The assay is based on the indication of the enzymatic consumption of H2O2 (HP), using a fluorescent europium-tetracycline (Eu3TC) complex as indicator. On addition of HP, this complex forms a highly fluorescent adduct (Eu3TC-HP), which is decomposed in the presence of POx to form the weakly fluorescent europium-tetracycline (EU3TC). Hence, the activity of the enzyme can be directly determined by means of the luminescent EU3TC complex as indicator. The POx assay demonstrated herein was elaborated starting from a spectral characterization of the complex systems involved. Due to the long lifetime of lanthanide luminescence, both steady-state and time-resolved luminescence assays can easily be performed. The time-resolved assay can quantify POx in the range from 4.0x10(-5) to 5.9x10(-3) U mL(-1), with a limit of detection of 1.0x10(-5) U mL(-1). The effects of POx inhibitors such as cyanide, hydroxylamine, and azide have also been studied. In addition, a time-resolved fluorescent detection method for a POx-based enzyme-linked immunosorbent assay (ELISA) has been developed, which is demonstrated in a sandwich model assay with bovine IgG serving as analyte. Furthermore, a time-resolved fluorescent imaging method is demonstrated that makes use of a straightforward imaging set-up adjusted to the optical properties of the europium reagent.