Interferon gamma (IFN-gamma) is a cytokine that is mainly secreted from immune cells. It plays an important role in regulating neighboring cellular activities through interactions with cell membrane proteins. Because of its biological importance, there has been increasing demand for a biosensor that can respond to IFN-gamma. Here, we report a fluorescent aptamer sensor that targets IFN-gamma. The 25-nt IFN-gamma aptamer was converted into a turn-on fluorescent sensor by labeling the 3'-end with fluorescein. The designed sensor achieved fluorescence sensing of IFN-gamma with nanomolar affinity.