A simple sensitive spectrofluorimetric method was developed for the determination of metacycline. Ethyl substituted β‐cyclodextrin (DE‐β‐CD) can remarkably enhance the fluorescence intensity of the metacycline (MC)–Eu3+ complex at λ = 612 nm and the enhanced fluorescence intensity of Eu3+ ion is in proportion to the concentration of MC. Optimum conditions for the determination of MC were also investigated. The linear range and the detection limit of this method for the determination of MC are 2.20 × 10−7–1.47 × 10−5 mol/L and 3.60 × 10−8 mol/L, respectively. The recommended method is practical and relatively free interference from coexisting substances and can be successfully applied to determination of MC in samples of serum and urine. The interaction mechanism between the complex MC–Eu3+ and DE‐β‐CD has been studied. The enhancement of the fluorescence at λem = 612 nm of Eu3+ ion is because of the inclusion complex MC–Eu3+–DE‐β‐CD.