The invention provides a high-performance liquid detection and content determination method for ectoine. Chromatographic conditions are as follows: the chromatographic column adopts an adamantine-bonded chromatographic column; the detector adopts a DAD detector; the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is methanol; the mobile phase B is an aqueous buffersolution of monopotassium phosphate; and the elution mode adopts isocratic elution. By utilizing the chromatographic conditions, a test solution and a standard solution are injected into a liquid chromatograph, the chromatogram is recorded, the peak area is calculated according to an external standard method, and the content of ectoine in the sample can be accurately determined. The method disclosed by the invention is short in detection time, excellent in separation effect, high in precision and accuracy and low in cost and has an important application value in aspects of strain screening and related quantitative researches during ectoine production.