NaOH pretreatment is a convenient and effective method which is widely used in rice straw anaerobic digestion. But the mechanism of the alkaline (NaOH) hydrolysis of biopolymers compositions and polymeric cross-linked network structures of rice straw cell wall need further study. This paper firstly studied the effect and mechanism of alkali pretreatment on anaerobic digestion and biogas production of rice straw by using a combination of confocal Raman microscopy and transmission electron microscope. First, the original rice straw and the rice straw pretreated by NaOH were taken for mapping scanning by confocal Raman microscopy with micron-scale spatial resolution. Then principal component analysis was adopted to extract main information of Raman spectra, it could be found that the two types of samples were respectively presented with ray-like distribution in the first two principal component space, which were with cumulative contribution of 99%. And there was a clear boundary between the two types of samples without any overlapping, indicating that there was a significant difference of Raman spectral characteristic between original rice leaf and rice leaf pretreated by NaOH. Further analysis of the loading weights of the first two principal components showed that the Raman peaks at 1 739, 1 508 and 1 094 cm-1 were the important bands, and these three Raman peaks were attributed to the scattering of hemicellulose, cellulose and lignin respectively. Following, chemical imaging analysis of hemicellulose, cellulose and lignin were achieved by combining these Raman peaks and microscopic image information. It could be found that the NaOH pretreatment resulted in a loss of dense spatial uniformity structure of tissue and great decreases of the contents of these three ingredients, particularly lignin. It can be concluded that it is feasible to non-destructively measure hemicellulose, lignin and cellulose in rice straw tissue by confocal Raman microscopy, and to achieve chemical imaging analysis of the three ingredients in tissue, and this research will be much help for revealing the promotion mechanism of NaOH pretreatment for the rice straw fermentation and biogas production.