The development of real-time single-molecule detection and tracking technology in time and space for multiple bio-molecules in intact cells has important significance for the study on molecular behaviors in life processes. However, limited depth of field is the main drawback of conventional microscopy that prevents observation and tracking of multiple molecules in intact cells. Based on the principle of wavefront coding, the design and fabrication of a novel diffractive optical element (DOE) which combines a distorted diffraction grating with a double-helix point spread function (PSF) phase plate so that it can simultaneously perform multi-plane imaging with double-helix PSF and realize an extended depth of field is presented. Experiments have been carried out on a self-built microscopic system based on the novel DOE, showing that a depth of field can be up to 12 μm. Experimental results are in good agreement with the theoretical predictions, thus proving the feasibility of this method.