This study aimed to develop bio-based polyurethane (PU) resin derived from tannin for the impregnation process of ramie fibers (Boehmeria nivea L.) and to investigate the mechanical and thermal properties of impregnated fibers. Tannin was extracted from Acacia mangium bark using hot-water extraction. The polymeric diphenylmethane diisocyanate (pMDI) was added into tannin extract to prepare tannin-based PU (TPU) resin. Degummed ramie fibers were impregnated by TPU resins for 1, 2, and 3 h at a temperature of 25 °C under a pressure of 50 kPa in a vacuum chamber. The weight gain of fibers after impregnation increased by 73.26%. After the impregnation process, the formation of urethane linkages on fibers was detected by fourier-transform infrared spectroscopy (FTIR) and micro confocal Raman spectroscopy. Field emission scanning electron miscroscopy (FE-SEM) coupled with energy dispersive x-ray (EDX) detected the TPU on the surface of ramie fibers, indicating by increasing of Nitrogen content on ramie fibers which derived from TPU. Using thermogravimetric analysis (TGA), the thermal stability of fibers increased from 14.2% to 32.6% of weight residue after heating at 750 °C. In addition, x-ray diffractogram (XRD) analysis showed that impregnation process increased the crystallinity of ramie fibers, resulting in increased of modulus of elastictiy by maximum of 29.5% and tensile strength by 4.7%, respectively, after 1 h of impregnation time. This study demonstrated the positive effect of impregnating ramie fibers by TPU resin on their thermal and mechanical properties, enhancing their more comprehensive industrial application as a value-added functional material.