This study evaluated the effects of different drying techniques on the physicochemical properties of Pleurotus citrinopileatus Singer (P. citrinopileatus), focusing on the ergothioneine (EGT) contents. The P. citrinopileatus was subjected to natural ventilation drying (ND), freeze-drying (FD), and hot-air drying (HD). EGT was extracted using high-hydrostatic-pressure extraction (HHPE), and response surface methodology (RSM) was employed with four variables to optimize the extraction parameters. The crude EGT extract was purified by ultrafiltration and anion resin purification, and its antioxidant activity was investigated. The results showed that the ND method effectively disrupted mushroom tissues, promoting amino acid anabolism, thereby increasing the EGT content of mushrooms. Based on RSM, the optimum extracting conditions were pressure of 250 MPa, extraction time of 52 min, distilled water (dH2O) as the extraction solvent, and a 1:10 liquid–solid ratio, which yielded the highest EGT content of 4.03 ± 0.01 mg/g d.w. UPLC-Q-TOF-MSE was performed to assess the purity of the samples (purity: 86.34 ± 3.52%), and MS2 information of the main peak showed primary ions (m/z 230.1) and secondary cations (m/z 186.1050, m/z 127.0323) consistent with standard products. In addition, compared with ascorbic acid (VC), EGT showed strong free radical scavenging ability, especially for hydroxyl and ATBS radicals, at more than 5 mmol/L. These findings indicate that the extraction and purification methods used were optimal and suggest a possible synthetic path of EGT in P. citrinopileatus, which will help better explore the application of EGT.