Browning is one of the key factors that influenced the callus subculture of tree peony (Paeonia suffruticosa Andr.). Effects of medium composition and exogenous hormones: macro elements of Murashige and Skoog (MS salts) and iron salt (Fe2+), pH, agar and 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid (NAA) and kinetin (KT) on the callus browning of P. suffruticosa ‘Shan Hu Tai’ in vitro were studied in this paper. Results showed that the browning of P. suffruticosa callus were more sensitive to KT than 6-BA in different concentrations of 6-BA and KT separately with different concentrations of NAA, and reduced to the lowest (13.3%) under 0.5 mg·L-1 NAA plus 0.3 mg·L-1 KT. 1/4 × MS plus 1/4 × Fe2+ was the best basic medium in which the browning rate was only 18.2%. The browning rate of the callus was the lowest of 4.0% under pH 6.5 and the callus grew better in 7.0 g·L-1 agar than others. This study indicated that the best medium preventing P. suffruticosa callus in vitro from browning was: 1/4 × MS medium supplemented with 6.95 mg·L-1Fe2+, 0.3 mg·L-1 KT, 0.5 mg·L-1 NAA, 6.0 g·L-1 agar and 30 g·L-1 sucrose in pH 6.5.