Chaetominine (CHA), a novel framework tripeptide alkaloid, imparts an attractive cytotoxic against the human leukemia cell line K562, which is produced by Aspergillus fumigatus CY018. However, its pharmacological research is restricted by low yields in submerged culture, which needs to be resolved immediately by biotechnology. In this work, a co-addition strategy was applied to promote CHA production based on related inhibitors' addition and precursors' addition, inspired by the biosynthetic pathway analysis of CHA. CHA production reached 53.87mg/L by addition of 10mM shikimate, 10mM anthranilate, 20mM tryptophan, and 10mM alanine in shake flask. Compared to the control without addition of precursors, the activity of 3-deoxy-arabino-heptulosonate-7-phospahte (DAHP) synthase was significantly improved and the transcription levels of critical genes in shikimate pathway were up-regulated responded to the co-addition of precursors. The improvement of CHA production by co-addition of precursors was also successfully reproduced in the lab-scale bioreactor (5-L) system, in which CHA production reached 46.10mg/L. This work demonstrated that precursors' co-addition was an effective strategy for increasing CHA production, and the information obtained might be useful to the further improvement of CHA on a large scale.