Hypothesis: Emulsion droplets are versatile tools in chemical and biological fields and the separation of emulsion droplets is one of the most crucial steps in research and industry. Experiments: This work developed a nano-orifice based DC dielectrophoretic (DEP) system for the sorting of oil-in-water emulsion droplets by size and content in a microchannel. Then, the size-dependent separation of oil droplets was conducted and verified by the prediction of theoretical simulation. In addition, by selecting the suspending solution with a specific electrical conductivity, the oil droplets with similar sizes but different contents were separated based on their opposite DEP behaviors. Findings: The size-dependent separation of smaller silicone oil droplets (7.5 and 11 mu m in diameter) with a high separation resolution, i.e., size difference of only 3.5 mu m was achieved and showed good agreement with the simulation results. The positive and negative DEP behaviors of the droplets varying with the electrical conductivity of the suspending solution were discussed and different types of droplets of similar sizes but different contents were separated. This paper presents the DC-DEP method based sorting of emulsion droplets by size and content in nanofluidic chip for the first time, providing a platform to manipulate individual emulsion droplet. (C) 2017 Elsevier Inc. All rights reserved.