Molecular structure changes are closely related to nutrient utilization and availability. However, so far little research was found to determine the processing induced changes on carbohydrate structure in co-products from bio-oil processing. The objectives of this study were to investigate synergistic effects of conditioning temperature (70, 80 and 90°C) and time (50 and 75 s) during the pelleting process on carbohydrate structure profile of the co-products from bio-oil processing (canola meal). The vibrational molecular spectroscopy (ATR-VMS) with chemometrics was used to determine the impact of pelleting at different conditions on the inherent molecular structure changes. The molecular spectral analyses, Univariate and Multivariate spectral analyses, were used in this study. Multivariate spectral analyses included cluster analysis and principal analysis. The chemical functional groups mainly associated with carbohydrate structure profile in this lipid-free co-products (or with very little lipid) included cellulosic compounds (CEL, ranged at ca. 1302–1186 cm⁻¹), structural CHO (SCHO, ranged at ca. 1488–1186 cm⁻¹) and total CHO (TCHO, ranged at ca. 1193–879 cm⁻¹). The results showed that the pelleting process was able to alter inherent structures of CHO functional groups in the co-products from bio-oil processing. The univariate molecular analysis indicated that spectral intensities of CHO functional groups in the co-products were significantly affected by the pelleting process in the current study ( P < 0.05 ). Altering processing conditions resulted changes in molecular structure features of CHO functional groups except TCHO. The results of multivariate spectral analysis of CHO indicated that inherent CHO structural characteristics of all functional groups were not fully distinguished. This study demonstrated that the pelleting process under the conditions investigated caused partial changes in carbohydrate structures in terms of the spectral features of specific functional groups. These changes were not sufficient enough to make the entire spectral region of CHO functional groups become fully distinguishable. Future research is needed to investigate the interactive relationships between the absorption intensities of carbohydrate functional groups (TCHO, SCHO, CEL) and biodegradation and digestion of the co-products in order to reveal how carbohydrate molecular structure changes induced by processing affect nutritive availability.