The thickness of the separator is an important factor affecting the microstructure, physical properties, and electrochemical performance of lithium-ion batteries. Here, 5, 7, 9, and 12 μm PE separators were compared and analyzed and the internal relationship between the structure and properties of the separator and the physical and electrochemical properties is discussed. Results show that the micropore density of the 9 μm separator is large and uniform, and the pore size of each micropore is also relatively large. With an increase in separator thickness, porosity increases gradually. The air permeability of the thicker separator is significantly better than that of the thinner separator. With an increase of the thickness of the separator, the maximum stress of the separator gradually decreases. The 5 μm separator showed the best thermal stability, followed by the 9 μm separator. Physical properties of the separator largely determine its electrochemical performance. The ionic conductivity and lithium-ion transference number of thicker separators are higher than those of thinner separators. By constructing the weighted performance index, we found that the comprehensive performance of the thicker separator is better than that of the thinner separator. However, considering the poor cycle stability of the 12 μm separator, this study believes that the 9 μm separator has the best comprehensive performance