The invention discloses a cell patterning structure based on an ordered polymer film, a preparation method and an application in cell proliferation inhibition, belonging to the technical field of cell positioning and assembling. The method comprises the following steps: preparing an ordered porous film the inner wall of which is loaded with a positive charge surfactant, selectively assembling polyelectrolyte with charges which is opposite to that of cells on the inner walls of pores of the ordered porous film, selectively adsorbing inside the pores through the cells, and the like. The cell patterning structure disclosed by the invention is simple and easily available, convenient to operate, gentle in condition, short in period and capable of achieving selective distribution of cells in the ordered honeycomb-shaped film pores, and moreover through selection of cells of appropriate sizes, a single cell array in which each pore only has one cell can be prepared. The single cell array has significant meanings in the fields such as biosensors, tissue engineering, single cell carrying and behavior operation. The structure has significant application values in aspects such as cell transferring and transportation, cell proliferation inhibition and treatment on cancer and other diseases.