Endophytic fungi can infect plant tissues without causing disease symptoms and show plants can grow well. One group of endophytic fungi is the dark septate endophyte (DSE) fungus. DSE fungi are reportedly capable of producing phytohormones such as IAA. In plants, the IAA hormone controls physiological processes and regulates cell elongation in stems and roots. This study aims to isolate, identify, and characterize IAA hormones DSE fungi produce from sunflower plants' roots. This research method includes measuring the optimization of IAA hormone production produced by endophytic fungi, including incubation time and the addition of L-tryptophan. In addition, IAA production analysis from DSE fungi was also carried out using thin-layer chromatography, adsorption chromatography, and high-performance liquid chromatography (HPLC). This study showed that DSE fungus isolates produced the highest IAA concentration with the addition of L-Tryptophan precursors as much as 0.5, which was 9.8 ppm, and the incubation time on the eighth day was 25.92 ppm. The results of the analysis of IAA hormone levels in growth media were obtained at 0.679 mgmL-1. There is still a minimal amount of phytohormone compounds obtained from isolation activities in this study, and it is necessary to find an appropriate isolation process so that the maximum amount of compounds is obtained to produce IAA.