[Objective] To explore the changes in the yield and composition of extracellular polymeric substances (EPS) ofAlternaria sp. CGMCC 17463, a strain of dark septate endophyte (DSE), cultured for different time periods. [Methods] We conducted the shake flask experiment to compare the yield, structure, composition, and activity of EPS synthesized by a DSE strain cultured for different time periods. [Results] From day 4 to 12, the growth of the DSE strain entered the logarithmic and stationary phases. During this period, the EPS synthesis rate was high, with the yield reaching 1.41 g/L on day 12. Afterwards, the EPS synthesis rate gradually decreased. The component analysis revealed that the extracellular polysaccharide content was the highest on day 12 in the EPS samples of equal mass. As the growth of DSE continued and entered the decline phase, mycelial lysis occurred, significantly increasing the protein content in the EPS. Functional group analysis showed that as the incubation time was extended, the functional groups in the EPS presented changes only in the content but not species. The results of scanning electron microscopy and particle size analysis showed that the EPS composition gradually changed with the increase in incubation time. Specifically, the EPS components with the particle size smaller than 5 μm presented increased volume percentage, while those with the particle size larger than 100 μm showed gradually decreased volume percentage. Furthermore, the EPS possessed the ability to scavenge oxygen free radicals and retain water, which were significantly influenced by the changes in EPS composition. [Conclusion] The day 12 marks the optimal time point for the production of EPS with high polysaccharide content, while the day 24 marks the optimal time point for the production of EPS with high protein content. This result establishes a foundation for the application of EPS in the complex eco-environment of mines.