An enzymatic assay for glucose based on the use of the fluorescent probe for hydrogen peroxide, europium(III) tetracycline (EuTc), is described. The weakly fluorescent EuTc and enzymatically generated H2O2 form a strongly fluorescent complex (EuTc-H2O2) whose fluorescence decay profile is significantly different. Since the decay time of EuTc-H2O2 is in the microseconds time domain, fluorescence can be detected in the time-resolved mode, thus enabling substantial reduction of background fluorescence. The scheme represents the first H2O2-based time-resolved fluorescence assay for glucose not requiring the presence of a peroxidase. The time-resolved assay (with a delay time of 60 mus and using endpoint detection) enables glucose to be determined at levels as low as 2.2 mumol L-1, with a dynamic range of 2.2-100 mumol L-1. The method also was adapted to a kinetic assay in order to cover higher glucose levels (mmol L-1 range). The latter was validated by analyzing spiked serum samples and gave a good linear relationship for glucose levels from 2.5 to 55.5 mmol L-1. Noteworthy features of the assay include easy accessibility of the probe, large Stokes' shift, a line-like fluorescence peaking at 616 nm, stability towards oxygen, a working pH of approximately 7, and its suitability for both kinetic and endpoint determination.