Intercalation of cytosine into Eu3+-doped hydrocalumites and the fluorescence of the Eu3+-doped hydrocalumite response to cytosine has been investigated. XRD patterns showed that the basal spacing of Eu-doped hydrocalumite obviously increased after it exposed to various content of cytosine, revealing the intercalation of cytosine into Eu3+-doped hydrocalumite. TG-DSC curves and IR spectra of the intercalated samples are different from that of the Eu-doped hydrocalumite and cytosine, indicating the interaction between the Eu3+-doped hydrocalumite and cytosine. Fluorescent spectra suggested that the fluorescent changes of Eu3+-doped hydrocalumite depended on the concentration of cytosine solution. This fluorescent change would be potential application in biological probe in view of the biocompatibility of Ca2+ ions and the fluorescence of Eu3+ ions. Moreover, the Eu3+-doped hydrocalumite would be a healthy and cheap fluorescent material applied in biology or healing drugs fields.