Provided are a five-chamber nucleic acid extraction device and method based on immiscible phase interfacial tension. The device comprises a sample chamber, an outlet of the sample chamber is sequentially connected with a first immiscible phase chamber, a washing chamber, a second immiscible phase chamber and an elution chamber through trapezoid micro-channels, and a magnet is arranged on the outerportions of the sample chamber, immiscible phase chambers, washing chamber or elution chamber; the method comprises the steps that a biological sample, a cell lysis solution and a nano-magnetic beadare injected into the sample chamber, firstly, an elution buffer solution is added into the elution chamber, then a washing solution is added into the washing chamber, and lastly the two immiscible phase chambers are filled with immiscible phases; the magnet rotates at the outer side of the bottom of the sample chamber, so that the nano-magnetic bead reacts with the biological sample to form a magnetic bead-nucleic acid compound, the magnet moves to the first immiscible phase chamber, the washing chamber, the second immiscible phase chamber and the elution chamber in sequence, so that the magnetic bead-nucleic acid compound enters the elution chamber for elution to release a nucleic acid. The five-chamber nucleic acid extraction device and method based on the immiscible phase interfacial tension have the advantages that the rapid and convenient extraction of the nucleic acid is achieved, the consumption of the reagent sample is reduced, and the loss of the nucleic acid during the extraction is reduced.