Aiming at the ergonomic problem of operator fatigue in assembly line, a method of human fatigue analysis and improvement based on Metabolic Energy Expenditure (MEE) was investigated. On the basis of ergonomic simulation technology, the method introduces MEE to quantitatively evaluate operator fatigue in assembly work. Human fatigue analysis and improvement in assembly work can be accomplished through the decomposition and the parameter acquisition of operator's motion, the evaluation and calculation of human fatigue based on MEE, the proposal of human fatigue improvement scheme, and its simulation verification and implementation. The method's validity and feasibility were verified by its successful application in the tire assembling process of an automotive general assembly plant.