Abstract: Preparation and expansion methods for human pluripotent stem cell-derived human retinal pigment epithelial cells are provided. The preparation method includes the following steps: collecting 3D-PRE spheroids derived from human pluripotent stem cells, performing mechanical separation to remove non-RPE cells and clusters which are non-pigmented and retain a pigmented RPE cell sheet, enzymatically digesting the pigmented RPE cell sheet to obtain an RPE single cell suspension, and thereby the human pluripotent stem cell-derived human retinal pigment epithelial cells are obtained. The expansion method includes centrifuging the RPE single cell suspension and removing a supernatant, resuspending in an RPE cell medium and seeding into a cell culture container precoated with extracellular matrix to allow primary culture, and subculturing the cells after cells spread out.