Due to cell heterogeneity, the differences among individual cells are averaged out in bulk analysis methods, especially in the analysis of primary tumor biopsy samples from patients. To deeply understand the cell-to-cell variation in a primary tumor, single-cell culture and analysis with limited amount of cells are in high demand. Microfluidics has been an optimum platform to address the issue given its small reaction volume requirements. Digital microfluidics, which utilizes an electric signal to manipulate individual droplets has shown promise in cell-culture with easy controls. In this work, we realize single cell trapping on digital microfluidic platform by fabricating 3D microstructures on-chip to form semi-closed micro-wells. With this design, 20% of 30 x 30 array can be occupied by isolated single cells. We also use a low evaporation silicon oil and a fluorinated surfactant to lower the droplet actuation voltage and prevent the drop from evaporation, while llowing cell respiration during the long term of culture (24 h). The main steps for single cell trapping on digital microfluidics, as illustrated in this protocol, include 3D microstructures design, 3D microstructures construction on chip and oil film with surfactant for single cell trapping on chip.