Graphene is a planar two-dimensional material composed of sp²-bonded carbon atoms with extraordinary electrical, optical and mechanical properties, and considered as one of the revolutionary electronic component materials in the future. Some studies have shown that the inert gas ion irradiation as a defect introducing technique can change the structure and properties of graphene without introducing additional effects. In this paper, the 5.4 keV He ion irradiation at the dose ranging from 0.7 × 10¹³ cm^–2 to 2.5 × 10¹³ cm^–2 has a strong effect on graphene deposited by CVD technology. The X-ray photoelectron spectroscopy (XPS), Raman spectroscopy (Raman) and semi-conductor parameter analysis instrument are used to study the changes in the microstructure and electrical properties of graphene before and after irradiation. Detailed analysis shows that the defect density increases gradually with the irradiation dose increasing. Raman spectrum shows that when the irradiation dose increases to 1.6 × 10¹³ cm^–2, the value of I_D/I_G begins to decrease, and XPS shows that the irradiation changes the structure of C chemical bond in graphene which causes the bonding state of C—C sp² to be destroyed and partly converted into the C—C sp³ bonding state. Therefore, the structure of graphene begins to transform from nano-crystalline structure into sp³ amorphous structure. Simultaneously, increasing defects causes the graphene conductivity to continuously decrease, and also gives rise to the electrical transition from defect scattering mechanism based on Boltzmann transport to the hopping transport. The positive voltage direction offset of V_dirac increases nearly in direct proportion, which is due to the enhancement of graphene’s p-type doping effect caused by defects and adsorbed impurities. This work conduces to the understanding the mechanism of He ion interaction with graphene, and also provides an effective way of controlling the electronic properties.