Although many efforts have been made to engineer cell sheets with anisotropic patterns, current techniques still exhibit several shortcomings including uncontrolled harvest time and deformation of pattern by contraction. In this study, we report a simple method to harvest a cell sheet with a striated structure of extracellular matrix (ECM) and myoblasts using a thermosensitive hydrogel micropatterned with different sizes (25 and 80 μm). The hydrogel supported the formation of a confluent monolayer of myoblasts with aligned morphology. When the temperature was reduced from 37 to 4 oC, the size of the hydrogel increased by approximately 1.2-fold within 10 min. In response to this change, a cell sheet was harvested and could be transferred to the desirable substrate through conformal contact between the hydrogel and target. We further examined the effect of pattern size on alignment of ECM/cell assembly by fast Fourier transform (FFT) analysis of fluorescently stained stress fibers and ECM proteins within the harvested cell sheet. The cell sheet maintained alignment of confluent myoblasts after being harvested to the substrate. In addition, the topologic patterns also promoted formation of aligned myotube on the harvested cell sheet, which was important for muscle tissue regeneration. However, the pattern size appeared to have no influence on alignment of cells on the cell sheet. Finally, a bi-layered structure was fabricated in which each layer was stacked with aligned direction on the cell sheet being perpendicularly assembled. Collectively, our platform could be used for rapid harvest of cell sheets with aligned architecture of ECM/cell, which can be applied to fabrication of welldefined 3D tissue for tissue engineering.