This study introduced the research and development of a portable and miniaturized system for the measurement of the refractive index of sub-microliter liquid based on a microfluidic chip. A technical method of double-beam interference, was proposed for use in the measurement. Based on this, by using a laser diode as a light source, changes in the refractive index were calculated by utilizing a complementary metal–oxide–semiconductor to detect the movement of interference fringes of the liquid. Firstly, this study simulated the effects of influencing factors on the interference infringes of two Gaussian beams, such as their spot sizes, distance between two beam spots, and detection range. Secondly, this research introduced the system design and construction of the double-beam interference method and analyzed the results of refractive index tests on sub-microliter aqueous glucose solutions with different concentrations. The measurement accuracy reached 10 ⁴ refractive index units. This system has a compact structure and is rendered portable by using batteries for its power supply. The entire system is designed to be a double Z-shaped structure with a length of about 15 cm, a width of 5 cm, and a height of about 10 cm. It can be used to measure changes in the refractive index of sub-microliter to nanoliter liquids based on the use of a microfluidic chip.