We propose a new microfluidic device that can be used to determine the change in the negative dielectrophoresis (n-DEP) of dielectric microbeads when a small amount of DNA is attached to them. We previously proposed a DNA detection method based on changes in the DEP of microbeads induced by the attachment of DNA. When target DNA is attached to the microbeads having n-DEP property, the DEP changes from negative to positive. This occurs because electric charges of the DNA increase the surface conductance of the microbeads. Thus, only the DNA-labeled microbeads are attracted to a microelectrode by positive DEP. The trapped DNA-labeled microbeads can be counted by dielectrophoretic impedance measurements. A large amount of DNA (approximately 105 DNA molecules) is required to change the DEP from negative to positive. Even though this method can be combined with DNA amplification, reducing the amount of DNA required can help us to shorten the reaction time. In this study, we aimed to detect DNA less than 105 DNA molecules by determining the change in the n-DEP change. To achieve this, we proposed a simple microfluidic device consisting of a single microchannel and a single pair of microelectrodes. Numerical simulations revealed that the device can identify the slight change in the n-DEP of the microbeads corresponding to the attachment of a small amount of DNA. In practical experiments, the fabricated device distinguished 10-1000 DNA molecules per microbead. This method represents a fast and easy method of DNA detection when combined with DNA amplification techniques.