The invention relates to a skate liver oil and a preparation method thereof. The skate liver oil is characterized in that the skate liver oil comprises 0.76-1.13mass% of alkoxy glycerin, 0.71-0.92mass% of squalene, 25-28mass% of EPA and DHA, 1200-1600IU/g of vitamin A, 120-150IU/g of vitamin D, and the other substances which are lipids. The preparation method comprises the following steps: freezing skate livers, unfreezing, chopping, carrying out enzymatic hydrolysis treatment, carrying out enzyme killing treatment after the enzymatic hydrolysis, centrifuging, freezing the obtained emulsion, unfreezing, demulsifying, centrifuging, refining, degumming by citric acid, deacidifying by a caustic soda-soda ash mixture (a mass ratio of caustic soda to soda ash is 2:1), carrying out low temperature treatment, and carrying out evacuating nitrogen-filling packaging. The method has the advantages of reasonable operation, low cost and mild conditions, and the obtained skate liver oil has a very low peroxide value, a very low acid value and no nutrition loss, is rich in vitamin A, vitamin D, EPA, DHA, and important squalene and alkoxy glycerin, has a reinforced healthcare effect, and is easy to preserve and circuit in the market because of the evacuating nitrogen-filling packaging.