Enzyme engineering is a combined technology of enzymology and engineering, which is becoming the major foundation for modern biotechnology and bio-economy in near future. Recently, a breakthrough in synthetic biology demonstrates a possibility to develop artificial biosystems for biofuel and biorefinery. As one of the cores in synthetic biology, enzyme engineering has drawn more attentions all over the world. To promote enzyme engineering research in China, invited reviews and selected research articles were published in this special issue of “Enzyme Engineering”. The reviews and research articles focus on the fields of discovery of new enzymes, mechanism of action modes, and production and application of enzymes.