The efficient conversion of biomass into biofuels is closely associated with the topochemistry of the cell wall. In this study, the topochemical correlation between carbohydrates and lignin in the Eucommia ulmoides cell wall was investigated in situ by confocal Raman microscopy. The carbohydrates and lignin were mainly collocated in the secondary wall of the fiber, ray parenchyma, and vessel in E. ulmoides. High carbohydrates were associated with low lignin or vice versa, indicating that a high concentration of carbohydrates leads to a drop in the degree of lignification. Furthermore, the band intensity ratio of S- and G-lignin to carbohydrates (I1333/I2889 and I1274/I2889) in morphologically distinct regions of fiber was calculated. In accordance with the wet chemical analysis, a higher ratio of lignin to carbohydrates was observed within the middle layer of the 3-year-old E. ulmoides fiber secondary wall. The results potentially extend the current understanding of the carbohydrate and lignin topochemistry in woody biomass and may facilitate an efficient wood bioconversion process in future biorefineries.