Abstract: OBJECTIVE To prolong the release time of 5-Fluorouracil(5-Fu) in vitro, β-cyclodextrin(β-CD) was used to functionalize graphene oxide(GO) and then loaded with 5-Fu. METHODS By using a modified hummers method to prepare GO, and the resulting GO could subsequently be esterified with β-CD to afford the β-CD functionalized GO(GO-β-CD). In addition, In vitro release of functionalized graphene oxide loading fluorouracil(GO-β-CD-5-Fu) could be determined by high performance liquid chromatography(HPLC). The structures and morphologies of GO, GO-β-CD and GO-β-CD-5-Fu were characterized by ultraviolet-visible(UV-vis) absorption spectroscopy, Fourier transform infrared(FTIR) spectroscopy, Transmission Electron Microscopy(TEM), Scanning Electron Microscopy(SEM) and Raman spectroscopy(Raman). RESULTS GO-β-CD were prepared and characterized successfully. GO-β-CD had a high drug loading capacity for 5-Fu, with a loading rate of 105%. The results of HPLC analysis showed the burst release effect of GO-β-CD-5-Fu was weakened in a gradient-release pattern,and the elimination half-life (T_1/2) was (4.5±0.08)d. CONCLUSION GO-β-CD was successfully loaded with 5-Fu and resulted in increase of the release time. Furthermore, GO-β-CD-5-Fu had a 324-fold longer T1/2 than 5-Fu.