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Two-dimensional (2D) nanomaterial-based membranes feature attractive properties for molecular separation and transport, which exhibit huge potential in various chemical processes. However, the low permeability and bio-fouling of the MXene membrane in water treatment become huge obstacles to its practical application. Herein, a highly permselective and anti-bacterial 2D nanofiltration membrane is fabricated by intercalating a polycation of polydiallyldimethylammonium chloride (PDDA) into the Ti₃C₂T_x MXene laminar architecture through a facile and patternable electrostatic assembly strategy. As a result, the as-fabricated Ti₃C₂T_x/PDDA composite membrane exhibits higher water permeance up to 73.4 L m⁻² h⁻¹ with a rejection above 94.6% for MgCl₂. The resultant membrane simultaneously possesses good resistance to swelling and long-term stability in water environments, even after 8 h. Additionally, the Ti₃C₂T_x/PDDA membrane also demonstrates a high flux recovery ratio of nearly 96.1% to bovine serum albumin proteins after being cleaned. More importantly, the current membrane shows excellent anti-adhesive and anti-microbial activity against Gram-negative <i>Escherichia coli</i> (<i>E. coli</i>) and Gram-positive <i>Staphylococcus aureus</i> (<i>S. aureus</i>), with inhibition rates of 90% and 95% against <i>E. coli</i> and <i>S. aureus</i>, respectively. This holds great potential for the application of the polyelectrolyte-intercalated MXene membrane in serving as a promising platform to separate molecules and/or ions in an aquatic environment.