This paper describes a novel real-time impedance chip for the detection of squalene-water emulsion phase separation. Each impedance chip contains eight pairs of indium tin oxide microelectrode arrays for detecting eight samples, and six chips can be connected with the switch relay to measure 48 samples in the system simultaneously. The proposed impedance chip has the advantages of needing only a small sample volume (0.5 mL), and provides parallel, continuous, and real-time detection. The effects of the surfactant concentration on the stability of a squalene/water emulsion were studied by means of a visual inspection, a conductance probe, and by impedance chip. Three different concentrations of Tween 20 surfactant (9, 17, and 29 wt%) were employed for the examinations. The results indicated that the phase separation rate was faster in the lower surfactant concentration. However, the emulsion of 29 wt% Tween 20 was fairly stable for more than 2 days since there were no signal changes according to the three detection methods. The reaction time (TR) for completing the measured phase separation process differed for each of the three methods (measuring aqueous phase height, conductance, and impedance, respectively). For the 9 wt% Tween 20, the reaction times were 24 h, 20 min, and 5 min in the tests using visual inspection, conductance probe, and impedance chip, respectively. For the 17 wt% Tween 20, the TR was also shorter when using the impedance chip method compared to the other two methods. Therefore the proposed impedance chip has a quick reaction response and provides an alternative and effective method to detect emulsion stability.