The development of high-performance polyamide nanofiltration (PA NF) membranes by introducing interlayers holds promise for efficient desalination. In this study, a novel PA NF membrane was prepared by incorporating the special biomass interlayer, which consisted of dehydration-modulated pomelo peel cellulose nanofiber (PCNF). Due to the removal of water, the proportion of exposed oxygen-containing functional groups within the PCNF interlayer increased significantly, and thus the diffusion of piperazine (PIP) could be slowed down effectively. Consequently, the diffusion-restricted PIP was enriched and anchored by PCNF and formed a PA layer with a unique layered structure in the interfacial polymerization (IP) reaction. The surface PA layer with lower a cross-linking degree could optimize water transport pathways and improve membrane permeability (28.67 LMH/bar). Meanwhile, the bottom PA layer, which was closely interspersed with PCNF, reduced pore size of the membrane and enhanced the rejection of Na2SO4 (98.63%). This study reveals a low-cost and eco-friendly strategy for preparing effective biomass interlayers, which provides a feasible approach for designing high-performance NF membranes.