Graphene oxide(GO)nanosheets possess several advantages,such as a large surface,outstanding biocompatibility,and straightforward chemical modification capability.They also have great potential as a drugcarrier.In this article,we radiolabeled GO nanosheets with99mTc,which satisfies the potential needs of microSPECT imaging probes in pre-clinical and clinical research.GO nanosheets were synthesized through the modified Hummers’method,then GO nanosheets with azide group covalently functionalized in two steps were conjugated to DOTA(1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid)and functionalized with an alkynyl group by means of click chemistry.Then through the addition and reduction of technetium-99m,the99mTc-DOTA-GO were attained.DOTA-conjugated GOs with lateral dimensions of 500–600 nm were synthesized.Both atomic force microscopy(AFM)and FT-IR were performed to characterize the GO-DOTA.Labeling efficiency of GO-DOTA with99mTc was>90%and radiochemical purities were>96%with purification.We successfully synthesized graphene oxide derivatives,DOTA-conjugated GOs,via Click Chemistry,and it was labeled with99mTc for SPECT imaging.High radiolabeling efficiency makes GO nanosheets suitable platforms for future molecular imaging research. Graphene oxide (GO) nanosheets possess several advantages, such as a large surface, outstanding biocompatibility, and straightforward chemical modification capability. They also have great potential as a drug carrier. In this article, we radiolabeled GO nanosheets with 99mTc, which satisfies the potential needs of microSPECT imaging probes in pre-clinical and clinical research. GO nanosheets were synthesized through the modified Hummers’method, then GO nanosheets with azide group covalently functionalized in two steps were conjugated to DOTA (1,4,7,10-tetraazacyclododecane-N, N, N-tetraacetic acid and functionalized with an alkynyl group by means of click chemistry. Through the addition and reduction of technetium-99m, the 99mTc-DOTA-GO were attained. DOTA-conjugated GOs with lateral dimensions of 500- 600 nm were synthesized.Both atomic force microscopy (AFM) and FT-IR were performed to characterize the GO-DOTA. Labeling efficiency of GO-DOTA with 99mTc was> 90% and radiochemical purities were> 96% wit h purification. We successfully synthesized graphene oxide derivatives, DOTA-conjugated GOs, via Click Chemistry, and was was labeled with 99mTc for SPECT imaging. High radiolabeling efficiency makes GO nanosheets suitable platforms for future molecular imaging research.