Cell sheet technology is applied to human articular chondrocytes to construct a tissue-like structure as an alternative treatment for cartilage defect. The effect of a gelatin manipulator, as a cell sheet transfer system, on the quality of the chondrocyte sheets was investigated. The changes of important chondrogenic markers and stress fibers, resulting from the cell sheet manipulation, were also studied. The chondrocyte cell sheets were constructed with patient-derived chondrocytes using a temperature-responsive polymer, and a gelatin manipulator as a transfer carrier. The properties of the cell sheets including sizes, expression levels of collagen type II and I, and the localization of the stress fibers were assessed and compared with those of the cell sheets harvested without the gelatin manipulator. Using the gelatin manipulator, the original size of the chondrocyte cell sheets was retained with abundant stress fibers and a decrease in the expression of collagen type II. Without the gelatin manipulator, although the cell shrinkage occurred, the cell sheet with suppressed stress fiber formation showed significantly high levels of collagen type II. These results supported that the changes of the stress fiber formation in the chondrocyte cell sheets affected the production of the chondrogenic markers. The densely packed tissue-like structure possessed a good chondrogenic activity, indicating its potential use in autologous chondrocyte implantation to treat cartilage defects. One of the challenges in cartilage tissue engineering is the maintenance of the chondrogenic activities during long-term culture. To overcome this problem, we proposed a new culture surface using the fifth generation dendrimer-immobilized surface (G5). Chondrocytes cultured on this surface formed various aggregates, providing desirable three-dimensional structure that closely mimicked the native environment of chondrocytes. As a result, higher chondrogenic markers (collagen type II and aggrecan) with lower de-differentiation marker (collagen type I) were observed in the cells on G5 surfaces as compared to those cultured on traditional polystyrene surfaces. This new surface would be beneficial for chondrocyte cell sheet engineering in our future study.