As an analogue of IDO1 (indoleamine 2, 3-dioxygenase 1), the well-known new therapeutic target, IDO2 is receiving increased attention. Herein, the expression and purification of recombinant human IDO2 (hIDO2) and the establishment of a hIDO2 bioassay system on both enzymatic and cellular levels are described. Nine tryptanthrin derivatives were screened for potential hIDO2 inhibitory activities, and their Ki values, enzymatic and cellular IC50 values, as well as the types of inhibition were measured. The typtanthrin derivatives 5i, 5c and 5d (especially 5i) were found to be potent hIDO2 inhibitors with superior efficiency far better than that of the most frequently-used inhibitor L-1-MT. Two ultimate purposes of the present study have been achieved: establishing an IDO2 bioassay system and screening novel IDO2 inhibitors that can be used (directly or with some modifications) for potential therapeutic applications.