Provided are a cell-holding substrate holder for preparing an observation specimen, and a cell-holding substrate kit, wherein the cell-holding substrate can be easily attached and detached. Also provided is a method of preparing an observation specimen, wherein cell organelles can be satisfactorily observed using an optical microscope, while three problems of cell detachment, drying, and loss of the three-dimensional properties can be simultaneously solved. The cell-holding substrate holder comprises (1) a support plate 1 having a cell-holding substrate arrangement portion 12 having a window portion through which water can pass; and (2) a removable sandwiching plate 2, 3 which has a window portion through which water can pass, and is capable of working in conjunction with the support plate 1 to sandwich and fix a cell-holding substrate 9 in the cell-holding substrate arrangement portion 12. The cell-holding substrate kit 10 comprises the cell-holding substrate holder and a cell-holding substrate 9. In the preparation method, cells are collected with an inorganic fiber aggregate, wet-fixed as they are, stained, and mounted with a mounting medium having a refractive index equivalent to that of the inorganic fiber.