An environmental and sensitive sample pretreatment method was established and combined with high‐performance liquid chromatography (HPLC) for the analysis of tetracycline hydrochloride (TC) in feed water and lake water. One element small molecule alcohol‐salt aqueous two‐phase system (ATPS) cannot effectively adjust the polarity of the system, but binary small molecule alcohol‐salt ATPS can adjust the polarity and improve the extraction efficiency of antibiotics. In this work, a binary ATPS based on ethanol +2‐propanol + (NH4)2SO4 system was formed and applied to the separation and purification of TC in real water samples. The influence factors on partition behaviors of TC were discussed, including the types and the concentration of phase salts, the volume ratio of alcohol, the pH value, extraction temperature, and the standing time. The response surface methodology was used to determine the best experimental conditions for multi‐factor experiments. Under this optimal condition, the extraction efficiency of TC reached 95.7%. This new method is considered to have significant application in the divorce of antibiotics.

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