Ergothioneine (EGT) is a natural antioxidant abundant in edible fungi. However, the low yield and extraction efficiency from natural sources of EGT limit its large-scale production. To improve the synthesis and extraction efficiency of EGT from Pleurotus citrinopileatus mycelium, a combination of single-factor tests and orthogonal experiments was employed in this study, using EGT biosynthesis yield as the index, the effects of culture environment and medium components on the fermentation process were systematically investigated. Meanwhile, key parameters of the extraction process such as solid-liquid ratio, extraction temperature, and extraction time were optimized with EGT extraction yield as the index. The results showed that the highest EGT yield of 16.89 mg·g-1 dw was achived when Pleurotus citrinopileatus mycelium was cultured under the conditions of temperature 27 ℃, rotation speed 210 r·min-1, liquid volume 100 mL/250 mL, initial pH value of 8.5, and the medium containing 2% mannitol, 0.3% casein peptone, 0.1% KH2PO4, 0.03% VB2 and 3 mmol·L-1 phenylalanine. Based on basic medium, under the conditions of solid-liquid ratio 1:30 (g:mL), extraction time 1.5 h, extraction temperature 60 ℃, and ethanol concentration 60%, the EGT extraction yield reached 1.64 mg·g-1 dw, and the solid-liquid ratio was identified as the most critical factor affecting extraction efficiency. Furthermore, this study first discovered that VB2 and KH2PO4 exert potential regulatory value on EGT biosynthesis. In conclusion, a highly efficient and synergistic fermentation and extraction process was established with stable and reliable parameters, which can provide a reference basis for the efficient preparation of EGT from Pleurotus citrinopileatus mycelium.