Biological channels have served as inspiration to design stimuli-response artificial nanopores. Here we propose an original approach to design a pH-gate nanopore based on polyethylenimine and chondroitin-4-sulfate (ChS) layer-by-layer self-assembly. This approach is interesting because it is rapid and permits monitoring in real time of functionalization. The study of ionic transport through these single nanopores reveals a selectivity on anions and pH-gate properties at low salt concentration. It is open at pH below 4 or 5 depending on salt concentration. These properties are explained by the modification of both charge and conformation of ChS as well as swelling of the polyelectrolyte complex.