Abstract A new spectrofluorimetric method was developed for determination of trace amount of Coenzyme II (NADP). Using europium ion-doxycycline (DC) as a fluorescent probe, in the buffer solution of pH 8.44, NADP can remarkably enhance the fluorescence intensity of the Eu 3+–DC complex at λ = 612 nm and the enhanced fluorescence intensity is in proportion to the concentration of NADP. Optimum conditions for the determination of NADP were also investigated. The dynamic range for the determination of NADP is 3.3 × 10 −7 to 6.1 × 10 −6 mol l −1 with detection limit of 6.8 × 10 −8 mol l −1. This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of NADP in synthetic water samples and in serum samples. Moreover, the enhancement mechanisms of the fluorescence intensity in the Eu 3+–DC system and the Eu 3+–DC–NADP system have been also discussed.