Objective To investigate the metabolic alteration of free Carnitine and Acylcarnitine profiles in premature neonates. Methods The dry blood spots samples were collected from 1,992 premature neonates and 4,035 full-term neonates delivered in our hospital from 2015 to 2016. The samples were subjected to analyze 31 kinds of Carnitine using the Tandem Mass Spectrometry. Results All carnitine except C10 and C10: 2 were changed significantly compared with that in full-term neonates. Compared with the full-term neonates, most of the mediumchain and short-chain acyl-carnitine were increased while most of the high-chain acyl-carnitine were decreased in the premature neonates. Conclusions Premature neonates exert totally different characteristic of carnitine metabolism; thus, proper nutrition strategies should be provided for the premature neonates during hospitalization.