Preparation of flexible, lightweight, stable, and robust electromagnetic interference (EMI) shielding materials is greatly desired but remains a scientific challenge. Herein, holocellulose nanofibers (HNFs) were employed to fabricate an alternating multilayered HNFs/MXene composite nanopaper with long-term durability and effective electromagnetic shielding performance by an alternate strategy. As a control, a hybrid method was adopted to prepare a nanopaper with a nacre-like lamellar structure. The alternating multilayered HNFs/MXene nanopaper with three layers of MXene (42.8% Ti 3 C 2 T x content) exhibits an impressive EMI shielding performance of 36.91 dB. More importantly, the mechanical properties of the alternating multilayered HNFs/MXene nanopaper are demonstrated at a superior level (tensile strength > 159.39 MPa, toughness > 7.36 MJ m −3 ). The outside HNFs and hexadecyltrimethoxysilane (HDTMS) hydrophobic layers of the alternating multilayered nanopaper can effectively protect MXene from oxygen and water molecules, decelerating the oxidation rate of MXene. Therefore, the EMI shielding efficiency of the alternating multilayered HNFs/ MXene nanopaper (the mass fraction of MXene is 40%) remained from 99.95 to 99.51% without obvious fatigue after 60 days in the atmosphere. This work provides an efficient strategy for promising materials for EMI shielding applications.