Ampicillin is a one of effective antibiotics against Gram-positive and Gram-negative bacteria. This study aimed to label ampicillin-loaded graphene oxide nanoflake (AMP-GO) with Tc-99m and evaluate of its in vitro binding to Staphylococcus aureus and Escherichia coli. Firstly, ampicillin was loaded into graphene oxide nanoflake prepared. AMP-GO was characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) techniques, and the amount of loaded ampicillin onto GO was determined by UV-Vis absorption spectroscopy. AMP and AMP-GO were labeled with Tc-99m using stannous chloride reducing agent. Labeling efficiency of Tc-99m-AMP-GO was found to be 97.66 +/- 2.06%. Tc-99m-AMP-GO has higher binding efficiencies to both S. aureus and E.coli than Tc-99m-AMP. Tc-99m-AMP-GO could be promising candidate as agent infection nuclear imaging. Furthermore, in vivo studies of Tc-99m-AMP-GO with infected rats are planned to be performed.