AbstractL-Carnitine is an endogenous substance essential for energy metabolism in mammals and a drug for the treatment of cardiovascular and neurological diseases. In this study, we reported and characterized a new impurity of L-carnitine (3-ethoxy-4-trimethylammonium 2-butenoate). The compound was synthesized from ethyl 4-chloroacetoacetate by a three-step reaction. It can be used as the impurity reference for the quality study and control of L-carnitine.