The large image area fluorescence microscopy imaging system can perform one-time imaging of large-size dPCR biological gene chips, but its depth of field (DOF, Depth of field) is small, which limits the object detection depth of the system. The wavefront coding technique is used to effectively expand the depth of field of the large image area fluo-rescence microscopy imaging system without reducing the resolution, which provides technical support for the subsequent one-time imaging of high-throughput multilayer stacked dPCR chips. However, considering that the forward design and simulation of the microscopic imaging system is not suitable for direct depth of field extension analysis,so it is inverted and flipped to analyze the focus depth extension range (i. e. , depth of field) on the image side (original object side). In order to determine the optimal phase plate parameter values, a genetic algorithm was written to link with ZEMAX's API interface, and the phase plate modulation coefficients were optimized, screened, and set in a dynamic interactive manner. A phase plate with a specific coefficient is added to the parallel light path in front of the aperture diaphragm of the large image area fluorescence microscopy imaging system to modulate the wavefront phase of the optical system, so that a consistent intermediate blurred image is produced within a range near the focus. The Wiener filter is used to restore the intermediate fuzzy image, and the simulation results show that the encoded image has a high resolution (up to 6. 5um) within the range of ±10 times depth of field of the traditional optical system, which can realize the depth of field extension of the system. The actual large image area fluorescence microscopy imaging system equipped with a phase plate has a good image restoration resolution (6.5 pm) within the range of ±7 times the depth of field.