The Eu(III) ternary complex with 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (H(3)DO3A) and picolinate "antenna" ligand ([Eu(DO3A)(L)](-)) reacts with carbonate anions to form the more stable non-fluorescent [Eu(DO3A)(CO3)](2-) complex. This substitution reaction was employed for indirect determination of carbonate formed in course of urea hydrolysis catalyzed by the urease enzyme. This new analytical procedure was proposed for the determination of both analytes (urea, urease) and this sensitive luminescence-based biosensor can be used in both kinetic time-dependent and equilibrium end-point modes. This method, with analytical figures of merit (limit of detection 0.05 mM, dynamic range 0.05-5.00 mM), does not show any systematic error and therefore can be utilized for urea analysis in biological samples (e.g., urine). It was also demonstrated that some metal ions (e.g., Zn(II), Pb(II), Cd(II), Ag(I), etc.) inhibit the enzymatic hydrolysis of urea and this effect can be utilized for their determination; therefore, it is recommended to add EDTA as masking agent prior to enzymatic urea analysis.