Objective To establish the cut-off value of amino acids, organic acids and aliphatic acids for newborn genetic metabolic disease screening in Ningxia, in order to provide theoretical basis for the laboratory diagnosis of neonate genetic metabolic diseases. Methods With non-derivative pretreatment of dry blood spots sample, tandem mass spectrometry method was adopted to detect amino acids, organic acids and aliphatic acids concentration in the whole blood samples of 45 602 neonates up to standard in Ningxia.Quality control products were from the two batches of kit and the U.S.centers for disease control.The percentile method was used to establish the cut-off value and identified P₁~P₉₉ as normal reference range.Wilcoxon rank sum test was performed to analyze the difference of cut-off value between Ningxia and Shanghai Xinhua hospital. Results Among 45 602 neonates who were screened,31 cases were diagnosed as postive,20 cases with HPA,1 case with MSUD,2 case with high methionine anemia,1 case with CTLN1,3 case with MMA,1 case with IVA,1 case with SCADD,1 case with MCAD,1 case with CPTII. Compared with the cut-off value of Shanghai Xinhua Hospital, the measurements of arginine,glycine,tyrosine,ornithine,leucine,valine,lsopentyl carnitine,twelve corbonyl carnitine,C0/(C16+C18) and so on were significautly different. Conclusion With the number of screening increasing and the range of screening expanded, more experience and positive predictive value will be accumulated to adjust the cut-off value of relevant measurements.