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There are a variety of amino acids and polysaccharides contained in <i>Pleurotus eryngii</i> that can improve immunity and prevent cancer in humans. The purpose of this research was to investigate the improved quality hypha of <i>P. eryngii</i>-3, which could shorten the ripening cycle of the hypha and increase economic benefits to industries. In this paper, the quality hypha of <i>P. eryngii</i>-3 was investigated for the DCW, total protein, polysaccharides, laccase activity, and so on. The medium was screened and carbon and nitrogen sources were screened and optimized as 40 g/L of corn flour and soybean meal by <i>P. eryngii</i>-3. Then, the effects of pH, temperature, volume, and rotation speed were investigated, in which the suitable fermentation conditions were, respectively, pH = 6, a temperature of 27 °C, 90 mL of fermentation broth, and a 150 r/min rotation speed. Meanwhile, the transcription levels of <i>Lcc1</i>, <i>Lcc5</i>, <i>Lcc8</i>, and <i>Lcc12</i> were upregulated by 10.25-, 3.32-, 2.33-, and 6.29-fold, respectively, after the optimization. In addition, the response surface method of the BBD was utilized for further improvements to the DCW by <i>P. eryngii</i>-3, in which the optimized DCW reached 17.2345 ± 0.7845 g/L.