Abstract: Currently,microscopic observation primarily relies on specific fluorescence staining, which can cause irreversible damage to samples and presents issues such as high cost, phototoxicity, and bleaching. This study proposes a novel method for mesoscopic 3D reconstruction without fluorescence, known as RGB-MesoLFM. A fluorescence-free labeled white light mesoscopic circuit is constructed, where the RGB three-band light field data are decoupled. Subsequently, based on fluctuating optics point diffusion functions, the point impulse response of the system at any depth within the three bands is computed. Finally, an objective function is developed using convex optimization theory, considering the RGB three-band light field data, point impulse response inputs, and iterative 3D deconvolution to reconstruct a high-resolution non-fluorescence labeled target object at any depth. The 3D reconstruction experiment is based on the light field sampling results of egg embryo slices, and can obtain slice images in the range of 0~−20 µm with a sequence interval of 4 µm. Compared with the traditional fluorescence reconstruction method, the method proposed in this paper does not require fluorescence, but uses white light for imaging. The reconstructed PSNR value of arbitrary depth slices is about 10% higher than that of traditional methods.