Abstract: The data was provided for assessing the accuracy and reliability by the proved method, and the uncertainty evaluation of L (-) -hydroxyproline in yoghurt was evaluated using spectrophotometry . The detection were developed repeatedly by the improved method referring to 《Guide to Uncertainty Evaluation in chemistry Analysis 》 and 《 Evaluation of Uncertainty in Chemical Analysis Measurement 》 , the uncertainty evaluation was completed by considering the factors for influencing the result and evaluation. The synthetically standard uncertainty of L (-) -hydroxyproline in yogurt was 0.0015g/100g , and expanded uncertainty was 0.0030g/100g. The improved method for detection L (-) -hydroxyproline was simple, effective, and the origin of final uncertainty evaluation was the content of L (-) -hydroxyproline in sample and the procession for sample.