We present a comprehensive study of alternating current cloud point extraction (ACPE) on a microchip. ACPE is an extraction technique for preconcentration of membrane‐associated biomolecules. To characterize and optimize ACPE, we carried out ACPE experiments under various experimental conditions including amplitude and frequency of applied voltages, flow velocity, and concentration of surfactant, analyte, and salt. We found that ACPE has an amplitude threshold (15 Vp–p), above which the extraction was more efficient. The dependence of the extraction on frequency (>5 MHz) was insignificant. Efficient extraction was achieved when the velocity of the test solution was 0.10∼0.67 mm s−1 and the concentration of surfactant was 0.10∼1.0%. In contrast, the extraction was independent of the concentration of analytes (0.20∼20 μmol dm−3). The technique was applicable to solutions with a salt concentration of 0.050∼0.15 mol dm−3 under temperature control of the devices. Solution temperature in ACPE was also studied. These results provide guidelines for use of the ACPE technique in microfluidic chemical and biochemical analyses.