Objective To investigate and analyze the effect of Taka-diastase used for the detection of vitamin B₂ （source of riboflavin-5'-sodium phosphate） on the results of the tested sample.Methods According to the requirements of the national standard method GB 5009.85—2016， Method One， the content of vitamin B₂ （source of riboflavin-5'-sodium phosphate） in the tested sample was determined to investigate the influence of Taka-diastase and its operational conditions on the quantification results.Results Taka-diastase from different sources had a major effect on the vitamin B₂ （source of riboflavin-5'-sodium phosphate） measurement results， and the maximum difference between the measured and theoretical values was more than 10 fold. The results indicated that an enzymatic hydrolysis time of 12 h， and pH 6.0—6.5 were optimal.Conclusion When the national standard method was used to detect vitamin B₂ （source of riboflavin-5'-sodium phosphate） in beverages， the selection of Taka-diastase had a significant effect on the detection results.