Graphene was first discovered in 2004 (Novoselov K S, et al. 2004 Science 306 666), it is a single atomic layer of sp2-bonded carbon atoms arranged in a honeycomb-like lattice. According to its extraordinary electronic, mechanical, thermal and optical properties, one can expect it to have a variety of applications in nanoscale electronics, composite materials, energy storage, and biomedicine fields. Although many experimental and theoretical studies on graphene have been carried, there still exist many obstacles to its applications. A representative example is nanoscale electronics (e.g., field-effect transistors and optoelectronic devices) that requires non-zero band-gap. Therefore, introducing defects into graphene and leading to band-gap opening are key steps for its technique applications.Recently, ion beam irradiation as a defects introducing technique was performed by Lee et al. (2015 Appl. Surf. Sci. 344 52) and Zeng et al. (2016 Carbon 100 16) through 5, 10, and 15 MeV protons and highly charged ions (HCIs) irradiating the graphene separately. Considering the advantages of simplity for preparing samples and feasibility in atmospheric condition of Raman spectroscopy compared with common characterization techniques (high resolution transmission electron microscopy, scanning electron microscopy, atomic force microscopy) for nano-materials, in both studies, Raman spectroscopy is used to obtain the evolution of ID/IG (ID is the peak intensity excited by defects, IG is the peak intensity origining from lateral vibration of carbon atoms) with different energies and fluences, respectively. In this work, considered are the following points:1) the absence of quantitive characterization for defects in the above two studies; 2) the low displacement energy of 25 eV required for a carbon atom to be knocked out (Zhao S J, et al. 2012 Nanotechnology 23 285703); 3) the complex interaction between HCIs and material. The irradiation effects of single layer graphene on silicon substrate are investigated by 750 keV and 1 MeV proton bombarding. This introduces the defects into graphene and thus leads to band-gap opening. By comparing Raman spectra of the samples before and after irradiation, a quantitive characterization about defects in graphene is achieved. Detailed analysis shows that 1) the value of ID/IG increases with the energy loss of incident proton, which is consistent with the result of SRIM simulation; 2) the average distance of defects LD increases with the incident proton energy; 3) the defect density nD decreases with the incident proton energy. These indicate that the damage effect for MeV protons in single layer graphene with substrate is similar to those in three-dimensional materials. The method presented here may facilitate the understanding of the physical mechanism of MeV proton interaction with two-dimensional materials, and provide a potential way of controlling the electronic structure and band-gap.