DNA separation is one of the fundamental procedures for gene analyses. In this study, we developed an electrophoresis microchip that can separate large size DNA (larger than 10 kbp). Our chip had an array of micro-fences in a channel and the separation was achieved based on the principle of size exclusion chromatography (SEC). The SEC-based separation has an advantage that the DNA molecules do not suffer large deformation during the migration in a molecular sieve. This is especially important for the separation of large DNA molecules since they can be easily ruptured by a mechanical stress. To achieve the separation, we conducted the model-based analysis and estimated the optimal electrophoretic voltages for the separation. Based on the estimated conditions, we succeeded in the separation of T4 DNA and λ DNA with our chip.