Dried blood spot (DBS) samples have been widely used in many fields including newborn screening, with the advantages in transportation, storage and non-invasiveness. The DBS metabolomics research of neonatal congenital diseases will greatly expand the understanding of the disease occurring and progression. In this study, we developed a fast liquid chromatography mass spectrometry-based metabolomics method for neonatal DBS analysis. More than 90% of the metabolites were stably detected within a single 3.175 mm-diameter disk compared with three disks of the same diameter. The influences of blood volume effects and chromatographic effects on metabolite levels were evaluated. The levels of 11.3% metabolites were different when 75 μL and 35 μL of blood volumes were used for DBS preparation. Chromatographic effects occurred in relatively large preparation volume with 6.19% metabolites having different MS responses when comparing central disks and outer disks from DBS prepared with 75 μL whole blood. The DBS storage stability study showed that in comparison to the benchmark (-80 °C storage), storing at 4 °C for 1 year had obvious influences on more than half metabolites. Storing at 4 °C and -20 °C for short term (4 and 14 days) and -20 °C for long term (1 year) had less influences on amino acids, acyl-carnitines and SMs, but affecting the stability of partial phospholipids. Method validation showed that this method has a good repeatability, intra-day and inter-day precision, linearity and recovery. Finally, this method was further applied to investigate metabolic disruptions of congenital hypothyroidism (CH). It was found that metabolic changes of CH newborns mainly involved in amino acid metabolism and lipid metabolism.