Provided is a novel method for producing a three-dimensional cell culture construct. A method for producing a three-dimensional cell culture construct including at least two or more laminated cell layers includes the following: seeding coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component; culturing the seeded coated cells in a culture medium; and using at least a portion of the culture medium continuously for 5 days or more. A method for producing a three-dimensional cell culture construct including at least two or more laminated cell layers includes the following: seeding coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component; and culturing the seeded coated cells in a culture medium. The culture process of the coated cells includes culturing the coated cells in 70 µL or more of the culture medium per 1 × 10 4 cells.
