The invention discloses a three-dimensional microscopic imaging method and system based on a light field microscopic system, wherein the method includes: S1, in the optical system, acquiring the first point diffusion of the three-dimensional sample from the object plane to the sensor plane of the main camera function and the second point spread function from the object surface to the secondary camera sensor plane, generate the first forward projection matrix and the second forward projection matrix according to the first point spread function and the second point spread function; S2, acquire the three-dimensional sample in The light field intensity image taken by the main camera in the optical system and the high-resolution intensity image taken by the secondary camera; S3, the light field intensity image and the first forward projection matrix, the high-resolution intensity image and the second The forward projection matrix performs 3D reconstruction to generate a 3D reconstruction result of the 3D sample. In this method, by adding one acquisition optical path, the signal-to-noise ratio of focal plane reconstruction is enhanced under the same number of iterations, and the reconstruction effect of light field microscopic imaging is greatly improved.