The variation in the microfibrils orientation of the Daemonorops jenkinsiana multilayered fiber and vessel was in-situ studied by polarized laser Raman spectroscopy with 532 nm exciting laser and a high-NA objective lens (125). Raman imaging obtained by integrating over the band range from 2 771 to 3 000 cm-1 displayed the morphologically distinct cell wall regions, including cell corner middle lamella, compound middle lamella and secondary wall. Furthermore, the fiber secondary wall displayed a concentric structure with alternating broad and narrow layers, while vessels had no obvious layering structure. Higher Raman C—O—C band (1 097 cm-1) intensity was visualized within the narrow layer of fiber wall from polarized laser Raman images, indicating the microfibrilsin these regions were more parallel to the incident laser electric vector and more perpendicular to the cell axis. The microfibrils orientation in vessel was uniform with in its secondary wall. Raman spectral analysis of different morphological areas of cell wall evidenced the band intensities of the C—O—C, CH and CH2 modes had a significant correlation with the polarization direction of incident light. When the orientation of cellulose microfibrils changed from parallel in respect to the fiber axis to with a high angle, the C—O—C signal reduced obviously. By comparison, the signal of CH and CH2 displayed slight decrease when changing the direction of incident laser, indicating the more sensitive nature of C—O—C vibration modes to polarization direction of incident light. Comparing the Raman spectra extracted from the narrow (Nl-R), broad layer (Bl-R) of fiber radial wall, as well as narrow (Nl-T), broad layer (Bl-T) of fiber tangential wall, it was found that the Nl-R had higher 1 097 cm-1 band intensity, while Bl-T displayed higher 2 897 cm-1 band intensity. Raman band ratio (I1 095/I2 897) can be used to predict microfibrils angle (MFA) in different cell wall types, qualitatively. The results showed that the ratio was highest in vessel secondary wall (132~110), followed by narrow layer of fiber secondary wall (092~055), and lowest in the broad layer of fiber secondary wall (042~033), indicating the highest MFA in the vessel secondary wall. This study provided a novel method and important theoretical guidance for the investigation on cell wall architecture, chemical composition distribution and micromechanics.