We herein report on a novel micro-refractometer with a self-calibration function. A micro-image defocusing (µ-ID) technique using optical path separation via a three-pinhole aperture is adopted to measure refractive index (RI). In order to achieve self-calibration, the microfluidic device is designed to have a self-calibration region into which reference fluids with known RIs are injected, as well as a measurement region into which a sample fluid is injected. In this way, the RI of the sample fluid is calculated automatically. In order to demonstrate the application of our proposed micro-refractometer, certified RI liquids with RIs of 1.300, 1.400, 1.450, 1.500, 1.550, 1.600, and 1.700 were used. The experimental demonstration described herein shows that the measured RIs have a deviation of less than 0.0009 with an uncertainty of ±0.001 refractive index unit (RIU) compared with their certified values.