Indirubin is an important natural substance and has positive effects on various diseases. However, the current process of producing indirubin is inefficient, making it difficult to produce indirubin of high purity; thus, it is commercially unavailable. In this study, a method of indirubin using non-recombinant Escherichia coil as a whole cell enzyme with indican as a substrate was developed. After confirming that indirubin was produced from indican by non-recombinant E. coil under general conditions, attempts to compare the yield and purity of indirubin were conducted under various pH, temperature and culturing media conditions. Under the optimum conditions, the yield was reliably determined to be about 25-35%, and it was further increased (1.8-2.1 fold) by replenishing the catalyst with freshly prepared whole cells. Since the established method was simple and reproducible, high purity indirubin would expected to be produced efficiently through improvement of whole cell enzymes and development of scale-up processes. (C) 2011 Elsevier Ltd. All rights reserved.