With the gradual rise of enzyme engineering, it has played an essential role in synthetic biology, medicine, and biomanufacturing. However, due to the limitation of the cell membrane, the complexity of cellular metabolism, the difficulty of controlling the reaction environment and the toxicity of some metabolic products in traditional in vivo enzyme engineering, it is usually problematic to express functional enzymes and produce a high yield of synthesized compounds. Recently, cell-free synthetic biology methods for enzyme engineering have been proposed as alternative strategies. This cell-free method has no limitation of the cell membrane and no need to maintain cell viability, and each biosynthetic pathway is highly flexible. This property makes cell-free approaches suitable for the production of valuable products such as functional enzymes and chemicals that are difficult to synthesize. This article aims to discuss the latest advances in cell-free enzyme engineering, assess the trend of this developing topical filed, and analyze its prospects. (C) 2020 The Chemical Industry and Engineering Society of China, and Chemical Industry Press Co., Ltd. All rights reserved.