At first, C-dots/manganese oxide nanocomposite was synthesized chemically. The presence of Mn3O4 nanostructures and C-dots in the synthesized nanocomposite was confirmed by using of XRD, EDS, FTIR and TEM. The proposed method for synthesis is easy and cheap. The Mn3O4/C-dots nanocomposite shows oxidase-like activity, which is higher than oxidase-like activity of Mn3O4 nanoparticles. In this manner, detection of ferrous ion was possible by using of a competitive reaction between 3,3′,5,5′-tetramethylbenzidine and Fe2+ ion in the presence of the synthesized nanocomposite. So, a sensitive and selective colorimetric sensor for determination of Fe2+ ion was introduced. The linear range and detection limit for determination of Fe2+ ion were obtained as 0.03–0.83 μM and 0.03 μM, respectively.