Development of environment-friendly fabrication processes is one of the requirements for the next-generation semiconductor technology. In this regard, organic materials in the nature have emerged as exotic semiconducting materials exhibiting biocompatibility, biodegradability, and sustainability. Among them, indigo has excellent semiconducting properties appropriate for application in field-effect transistors. However, fundamental understanding of its electronic structure, which is a crucial information to improve the electrical properties, is lacking. In this study, we investigated the electronic structure of an indigo film using in-situ ultraviolet photoelectron spectroscopy (UPS) and inverse photoelectron spectroscopy. The measured hole injection barrier (phi(h)) and electron injection barrier (phi(e)) of indigo on a Au substrate were equal, which is consistent with its ambipolar charge transport characteristic. To determine the minimum values of phi(h) and phi(e), the Fermi-level pinning at the indigo interface was analyzed. A series of UPS experiments were performed using substrates having different work functions. The slope diagram showed three regions typically observed at weakly interacting organic interfaces. The minimum phi(h) and phi(e) of indigo were measured to be approximately 0.90 and 0.25 eV, respectively. Based on this study, a robust strategy to improve the device performance could be established, which could expand the use of indigo to various applications.