The invention provides a lithium ion secondary battery as well as an isolating membrane and a preparation method of the isolating membrane. The isolating membrane comprises a porous substrate and a coating, wherein the coating is arranged on the at least one side of the porous substrate. The coating contains polyurethane with the structure shown in formula 1; the polyurethane is formed by a section A with isocyanate group at the tail end and a section B with amido at the tail end which have reaction between the isocyanate group and the amido so as to generate urea bond; in the polyurethane, m and n are integers which are larger than or equal to 1; the section A is generated by the reaction among monomers R1, R2 and R3; the section B is generated by the reaction between monomers R4 and R5. The lithium ion secondary battery comprises the isolating membrane. The isolating membrane has good physical and mechanical properties, high temperature resistance and adhesive property, and effectively improves the safety of the lithium ion secondary battery and solves the problems of the deformation of the lithium ion secondary battery.