Bioelectronic devices can be manufactured by organic-inorganic hybrid systems based on biomolecules and silicon semiconductors. The performance of the hybrid systems is largely determined by the adsorption manners of biomolecules on the silicon surface. In this paper, we demonstrated that the X-ray photoelectron spectroscopy (XPS) shake-up satellites and near-edge X-ray absorption fine-structure (NEXAFS) at the carbon K-edges can be used to distinguish the interface of guanine molecules anchored on Si(100) surface. There are only 9 possible stable guanine@Si(100) hybrid systems that have been found based on the density functional theory. According to the characteristic peaks, it is confirmed that NEXAFS spectra are more sensitive to the identification of adsorption configurations. While the first characteristic peak in the low energy region of NEXAFS is capable of distinguishing chemical bonds at the interface of the adsorption configurations. These results may facilitate a better understanding of the interface formations between biomolecules and silicon surfaces, which could be further utilized for the new bioelectronic device design.