Human intestinal organoids (hIOs) derived from human pluripotent stem cells (hPSCs) have immense potential as a source of intestines. Therefore, an efficient system is needed for visualizing the stage of intestinal differentiation and further identifying hIOs derived from hPSCs. Here, 2 fluorescent biosensors were developed based on human induced pluripotent stem cell (hiPSC) lines that stably expressed fluorescent reporters driven by intestine‐specific gene promoters Kriippel‐like factor 5 monomeric Cherry (KLF5mCherry) and intestine‐specific homeobox enhanced green fluorescence protein (ISXeGFP). Then hIOs were efficiently induced from those transgenic hiPSC lines in which mCherry‐ or eGFP‐expressing cells, which appeared during differentiation, could be identified in intact living cells in real time. Reporter gene expression had no adverse effects on differentiation into hIOs and proliferation. Using our reporter system to screen for hIO differentiation factors, we identified DMH1 as an efficient substitute for Noggin. Transplanted hIOs under the kidney capsule were tracked with fluorescence imaging (FLI) and confirmed histologically. After orthotopic transplantation, the localization of the hIOs in the small intestine could be accurately visualized using FLI. Our study establishes a selective system for monitoring the in vitro differentiation and for tracking the in vivo localization of hIOs and contributes to further improvement of cell‐based therapies and preclinical screenings in the intestinal field.—Jung, K. B., Lee, H., Son, Y. S., Lee, J. H., Cho, H.‐S., Lee, M.‐O., Oh, J.‐H., Lee, J., Kim, S., Jung, C.‐R., Kim, J., Son, M.‐Y. In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells. FASEB J. 32,111‐122 (2018). www.fasebj.org