The plant hormone auxin is a key morphogenetic signal that controls many aspects of plant growth and development. Cellular auxin levels are coordinately regulated by multiple processes, including auxin biosynthesis and the polar transport and metabolic pathways. The auxin concentration gradient determines plant organ positioning and growth responses to environmental cues. Auxin transport systems play crucial roles in the spatiotemporal regulation of the auxin gradient. This auxin gradient has been analyzed using SCF-type E3 ubiquitin-ligase complex-based auxin biosensors in synthetic auxin-responsive reporter lines. However, the contributions of auxin biosynthesis and metabolism to the auxin gradient have been largely elusive. Additionally, the available information on subcellular auxin localization is still limited. Here we designed fluorescently labeled auxin analogs that remain active for auxin transport but are inactive for auxin signaling and metabolism. Fluorescent auxin analogs enable the selective visualization of the distribution of auxin by the auxin transport system. Together with auxin biosynthesis inhibitors and an auxin biosensor, these analogs indicated a substantial contribution of local auxin biosynthesis to the formation of auxin maxima at the root apex. Moreover, fluorescent auxin analogs mainly localized to the endoplasmic reticulum in cultured cells and roots, implying the presence of a subcellular auxin gradient in the cells. Our work not only provides a useful tool for the plant chemical biology field but also demonstrates a new strategy for imaging the distribution of small-molecule hormones.