In the study, a suitable differentiation medium was obtained, using the callus of the healthy leaves of Euonymus europaea cultured under different conditions with different hormone ratios. The results showed that: the suitable medium for callus differentiation of Euonymus europaea was MS+1.0 mg·L^-1 6-BA+0.1 mg·L^-1 NAA; the callus of Euonymus europaea was suitable for culture under light with growth cycle of 28 days, and it would appear browning in different degrees after 28 days; the soluble sugar content in the callus rose firstly and then decreased; the activities of SOD and POD in the callus both showed two peaks, and the change trend of soluble protein was basically consistent with the POD activity; soluble protein played an important role in the callus differentiation, and the contents of soluble sugar and soluble protein, the activities of SOD and POD were higher grown under illumination than those under dark conditions. The activity of PPO decreased in the late stage of culture, which may be related to the browning of callus.