Abstract: The objective of this study was to understand tolerance and respond of Pleurotus nebrodensis to copper stress, and to offer a consultation for investigation to mechanisms of copper detoxification and for prevention of copper pollution. P. nebrodensis was incubated in submerged culture medium PDA, which containing different Cu²⁺: 0, 50, 100, 200, 300μmol/L. In hyphae of P. nebrodensis, the content changes of soluble sugar and proline were analyzed by anthrone colorimetry and by colorimetry, respectively. And the activity changes of peroxidase (POD) and catalase (CAT) were assayed by guaiaco method and by ultraviolet spectroscopy, respectively. The results showed that the content of soluble sugar and proline decreased sharply in hyphae with the concentration of Cu²⁺ increase in medium, and their content were 20.94% and 35.53% lower than the content of the contrast at Cu²⁺ 300μmol/L, respectively. However, the activity changes of POD and CAT were first increased and then had a decreasing trend with the concentration of Cu²⁺ increase, their maximum activities were at Cu²⁺ 200 and 100μmol/L, respectively. In response to copper stress, the activity changes of POD and CAT in hyphae of P. nebrodensis were very similar to that in advanced plant, only Cu²⁺ concentration were lower than that in advanced plant at their maximum activities. But the content changes of soluble sugar and proline were different from that of advanced plant, in which content of soluble sugar and proline showed a first increasing and then decreasing trend with the concentration of Cu²⁺ increase. Therefore, P. nebrodensis was sensitive to copper stress. And a suggestion is put forward that the degree of Cu²⁺ contamination was no higher than 100μmol/L for P. nebrodensis cultivation in environment.