Thin film composite polyesteramide nanofiltration membranes were fabricated with interfacial polymerization from carboxylated chitosan and trimesoyl chloride on a microporous polysulfone support membrane. Salt rejection was improved by building a multiple-layer structure that was formed by sequentially repeating the cycles of the interfacial reactions. The chemical structure, surface morphology and charge were characterized for the polyesteramide top layer. The effects of the concentrations of the reactant solutions and the number of cycles of reactant depositions and reactions on the separation performance were investigated. The single-layer polyesteramide membrane produced from 3.5 wt% carboxylated chitosan and 0.7 wt% TMC has a negatively charged surface and shows favorable separation performance: pure water permeation flux of 7.3 L/(m2 h) at 0.6MPa gauge feed pressure, and salt rejection of 95.0% for Na2SO4, 65.7% for MgSO4, 33.2% for MgCl2 and 66.3% for NaCl. The multiple-layer polyesteramide membranes show a more negatively charged surface and higher salt rejection than the single-layer polyesteramide membranes. The single-layer polyesteramide membrane PS-[(C-CS)1.0/TMC] with a relatively loose structure shows good retention for the reactive black 5 anionic dye. This study opens an interesting research area to explore a new type of thin film composite nanofiltration membrane.