A thickness evaluation method of the cell sheet according to the invention includes tomographically imaging a cell sheet by optical coherence tomography and obtaining a thickness distribution of the cell sheet based on a result of the tomography imaging. A tomographic image corresponding to one cross section of the cell sheet is obtained by tomography imaging while scanning the light in a main scanning direction. The tomography imaging is performed in every time while moving an incident position of the light at a predetermined feed pitch in a sub-scanning direction, thereby a plurality of the tomographic images corresponding to a plurality of cross-sections are obtained. One-dimensional thickness distributions of the cell sheet in the corresponding cross-sections are obtained based on each of the plurality of tomographic images, and a two-dimensional thickness distribution of the cell sheet is obtained by interpolating the one-dimensional thickness distributions.