Carbon microspheres, as a new type of carbon material, have a high specific surface area and high electrical conductivity. There are a lot of reactive groups such as hydroxyl carbonyl and carboxyl groups on the surface of the microsphere, which makes it suitable for a wide range of application prospects on adsorbent materials, catalyst carriers, electrode battery materials, et cetera. By this research, the hydrothermal carbon microspheres were synthesized from different carbon sources including the natural biomass (Vitis vinifera and Eupatorium adenophorum) and saccharides (xylose, glucose and sucrose). To explore the effect of the composition of the carbon sources on the morphology and chemical structure, the properties of different carbon microspheres were characterized by Field Emission Scanning Electron Microscope (FE-SEM), Attenuated Total Reflection Fourier Transform Infrared Spectrometer (ATR-FTIR) and X-ray Photoelectron Spectroscopy (XPS). The infrared spectra of 2 000~800 cm^-1 in the fingerprint characteristic region were selected. The original infrared spectra data were pretreated by automatic baseline correction, automatic smoothing, ordinate normalization and analyzed by principal component analysis (PCA) for probing the differences in spectral properties between different carbon microspheres. The results showed that the morphology and particle size distribution of carbon microspheres were closely related to the raw material. The carbon microsphere prepared by saccharides, which diameter was approximately in the 0.3~1.6 μm range, sucrose >glucose> xylose. And the particle size distribution of biomass carbon microsphere was 0.1~0.6 μm, Eupatorium adenophorum>Vitis vinifera. In the carbonation reaction, biomass and saccharides had a series of dehydration, decarboxylation, aromatization and condensation reaction. In terms of the chemical structure, no matter the complexity of carbon sources, the carbon microspheres all had aromatic ring structure and outer surface contained a high concentration of reactive oxygen groups, such as O—H, CHO. The PCA results showed that two main components represented 83.1% fingerprint region variable information, which could reflect the main information of the original spectrum. In the principal component scores figure, the distribution of different carbon microspheres was relatively independent, there were obvious differences in the chemical composition of the synthetic carbon microspheres derived from different carbon sources. Therefore, the difference in the intensity of absorption peak could provide an effective reference for the identification of carbon microsphere from different carbon sources. The results could provide a theoretical basis for further exploration of the spectral properties of carbon microspheres prepared by different carbon sources under hydrothermal conditions.