A multi-LED multiplexing 3D-FPM reconstruction algorithm based on a multi-layer diffraction model relates to the technical field of computational imaging, solves the problem that the acquisition rate is too slow when a plurality of LEDs illuminate, and comprises the following steps: step 1, constructing a loss function between a low-resolution picture obtained by the multi-layer diffraction model and a low-resolution picture actually acquired by a camera; step 2, updating a sample refractive index function of the thin sample layer by adopting a gradient descent method by utilizing the newest loss function, the newest incident light complex amplitude of the thin sample layer and the newest sample refractive index function of the thin sample layer, and updating a phase part of a newest pupil function by utilizing the gradient descent method; and step 3, judging whether the number of times of completing the step 2 reaches a preset number, if so, outputting a sample refractive index function, and otherwise, performing the step 1 again according to an updated result. According to the method, the number of LR pictures needing to be collected is greatly reduced, meanwhile, the influence of system aberration on a reconstruction result is effectively reduced, and the reconstruction precision of an algorithm is improved.