Investigation of the microscopic properties of cement concrete requires well-prepared samples with a flat and undamaged surface. The epoxy impregnation in concrete sample can prevent damage to the microstructure during polishing process for obtaining the flat surface. However, further exploration about the effect of epoxy impregnation on characterizing the properties of concrete has not been found. In this paper, the vacuum impregnation method is adopted to prepare the epoxy impregnated concrete and paste samples. The aspects of microscopic properties of impregnated and non-impregnated samples are evaluated and compared in terms of the surface elevation, the indentation modulus, and the thickness of the interfacial transition zone between the aggregate and the paste matrix (ITZ(agg-paste)) as well as that between the residual C3S clinker and the hydration product (ITZ(C3S-HP)). The techniques of optical microscope, backscattered electron microscope, scanning probe microscope (SPM) modulus mapping, and the instrumented indentation are adopted to measure the above microproperties. The results show that the epoxy impregnation is favoured in the microscope image analysis, nanoindentation measurement, and characterizing the ITZ(C3S-HP) by SPM modulus mapping. It is not recommended for characterizing the thickness of ITZ(agg-paste) by SPM modulus mapping and for measuring the micromechanical properties at the paste scale by microindentation.