Graphene oxide (GO) nanosheets possess several advantages, such as a large surface, outstanding bio-compatibility, 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 micro-SPECT 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,N0,N00,N000-tetraacetic acid) and functionalized with an alkynyl group by means of click chemistry. Then 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 synthe-sized. 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%with purification. We successfully synthesized graphene oxide derivatives, DOTA-conjugated GOs, via Click Chemistry, and it was labeled with 99mTc for SPECT imaging. High radiolabeling efficiency makes GO nanosheets suitable platforms for future molecular imaging research.