Natural fiber reinforced polymer-based composites have been growing into a type of green composites. The properties of natural fiber reinforced polymer-based composites are closely related to the structure of natural fibers. Bagasse fiber (BF) is one of the most used natural fibers for preparing natural fiber reinforced polymer-based composites. However, few examples of previous research touch on the quantitatively characterization of structure of BF and its effect on the properties of BF reinforced polymer-based composites. In this work, four kinds of BF including untreated BF (UBF), alkali treated BF (ABF), BF modified by silane coupling agent (SBF), and BF modified combining alkali treatment with silane coupling agent (ASBF) were prepared and melting blended with polylactic acid (PLA) to prepare PLA/BF composites. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), thermogravimetry (TGA) and mechanical properties testing were used to characterize and analyze the structure and properties of modified BF and its reinforced PLA-based composites. Results showed that the used methods changed the structure of BF and their bonding modes. The surface energies of UBF, ABF, SBF, and ASBF were 19.8 mJ/m², 34.7 mJ/m², 12.3 mJ/m², and 21.6 mJ/m², respectively. The O/C ratios of UBF, ABF, SBF and, ASBF are 0.48, 0.53, 0.47, and 0.51. Due to the synergistic effect of alkali treatment and silane coupling agent modification on the surface chemical properties, the content of silicon elements on the surface of ASBF (4.15%) was higher than that of ASBF (2.38%). However, due to the destroying of alkali treatment on the microstructure of BF, the alkali treatment had no prominently synergetic effect with coupling agent modification on the mechanical properties of PLA/BF composites. Alkali treatment removed the small molecular compounds from BF, decreased its thermal stability, and increased the crystalline region and crystallinity of cellulose. Meanwhile, alkali treatment made BF fibrillated and increased its contactable active area with the coupling agents, but destructed the nature structure of BF. The silane coupling agent played a more important role than alkali treatment did in improving the interfacial compatibility of PLA/BF composites.