Abstract: Objective To establish the stable FOXO1-GFP transfected U2OS cell line and detect U2OS-FOXO1-GFP indicative function of oxidative stress. Methods The FOXO1-GFP vector was transfected into U2OS cell. Stably transfected U2OS cell was screened using G418 and Flow Cytometer. After 4 hours treatment of AAPH, cell survival was tested by CCK-8 and the location of FOXO1-GFP was observed by fluorescence microscope. Results The stable FOXO1-GFP transfected U2OS cell line was successfully established. The percentage of FOXO1-GFP positive cells in total cells was 92% after screened through G418 and FCM. CCK-8 assay showed that when U2OS-FOXO1-GFP cells were treated with AAPH, cell survival dropped to 0.55±0.04 compared with control (P＜0.05). In control vehicle group, FOXO1-GFP was localized in the cytosol was observed by fluorescence microscope. When cells were treated with 40 mmol/L AAPH 4 hours, translocation of FOXO1-GFP to the nucleus was observed. Conclusion The U2OS-FOXO1-GFP cell line can indicate the oxidative stress. It would be a convenient tool to study the antioxidant responses drugs.