Objective To establish a rapid detection method for hydroxyproline in dairy using amino acid autoanalyzer. Methods Hydroxyproline in dairy hydrolyzate could react with ninhydrin and produce a yellow compound which has an absorption peak at 440 nm in the second channel of the instrument and comply with the Lambert Beer law within a certain concentration range. Citrate buffer solution as mobile phase, hydroxyproline and other 17 kinds of amino acids were separated at 440 nm quantitatively. Results The calibration curves showed a good linarity in the range of 0.05~1.0mmol/L(r=0.9999).The mean recovery was 91.5% with RSD 1.3%(n=5) and the limit of detection was 0.66 mg/L. Conclusion The method was rapid, convenient, accurate and reliable. It could be applied for quantitative detection of hydroxyproline in dairy.