In this study, we investigated the feasibility of enzymatic digestion of polysaccharides for cell sheet harvesting. Cellulose was digested using cellulase; in brief, cellulose was pre-coated under a confluent cell layer, and then enzymatic digestion of cellulose under the confluent cell layer enabled cell detachment with minimal cell damage, yielding cell sheets. For the surface adhesion of the cellulose, carboxymethyl cellulose (CMC) molecules were conjugated with dopamine (DA), and the synthesized CMC-DA was pre-treated onto the surface of the culture plates. Then, human mesenchymal stem cells (hMSCs) or corneal limbal epithelial cells (hCLEs) were cultured on the pre-coated CMC-DA and harvested using cellulase containing cell culture medium. Single hMSCs treated with cellulase showed higher proliferative activity, showing an aggregated morphology compared with trypsin-treated hMSCs. Additionally, hMSC sheets were detached from the pre-coated CMC-DA surface 10 min after cellulase treatment. Also, hCLE sheets were generated with a well-preserved morphology and transparency after cellulase-assisted cell sheet generation. These results demonstrate that the strategy of CMC-DA coating combined with cellulase enzymatic harvesting is an effective option for harvesting cell sheets.