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Background: Some fungal endophytes isolated from <i>P. ginseng</i> may present a new method of obtaining saponins. This experiment aimed to optimize the total saponin yield produced through in vitro fermentation by an endophytic fungus and analyze its saponin species in the fermented extract. Methods: Fermentation protocols were optimized with a uniform design and verified through regression analysis to maximize the total saponin yield. The saponin types under optimal fermentation conditions were then identified and analyzed using Liquid Chromatography–Mass Spectrometry. Results: The <i>Trametes versicolor</i> strain NSJ105 (gene accession number: OR144428) isolated from wild ginseng could produce total saponins. The total saponin yield could be increased more than two-fold through the optimization of fermentation conditions. The concentration of the total saponins achieved by the verified protocol 105-DP was close to the predicted value. The fermentation conditions of the 105-DP protocol were as follows: potato concentration 97.3 mg/mL, glucose concentration 20.6 mg/mL, inoculum volume 2.1%, fermentation broth pH 2.1, fermentation temperature 29.2 °C, and fermentation time 6 d. It was detected and analyzed that the fermented extract of 105-DP contained the ginsenosides Rf and Rb₃. Conclusion: The endophytic fungus <i>Trametes versicolor</i> strain NSJ105 has potential application value in saponin production.