<p indent="0mm">Plant fibers possess the advantages of green environmental protection and abundant sources. They exhibit specific mechanical properties comparable to man-made fibers and can become an important reinforcement of composites. Their unique hierarchical structures affect the load transfer mode of plant fiber reinforced composites (PFRCs). To serve the task of interfacial design of PFRCs, it is necessary to quantify their hierarchical interfacial properties. First, by applying the nanoindentation technology, the mechanical properties of the multi-layer interface of PFRCs, including the modulus, hardness, energy dissipation, and ability of crack initiation and propagation, were measured using the single-step and multi-step static nanoindentation methods. Subsequently, the fatigue characteristics of multiple interfaces in PFRCs under cyclic loading were evaluated via the nanoscale dynamic mechanical analysis. Finally, combined with the finite element simulation at the mesoscale, the multi-stage failure behavior of the hierarchical interfaces in the composites was presented by introducing multiple interface performance parameters obtained at the nanoscale. The results showed that the hierarchical structure of plant fibers was associated with the multi-stage failure behavior of PFRCs. The hierarchical interfacial failure caused by the crack initiation and propagation at the three types of interfaces occurred sequentially. The experimental results at the nanoscale provide support and basis for an in-depth analysis of the interfacial properties and failure mechanisms of composites on meso-microscale.