Paeonia ostii ‘Feng Dan’, a tree peony, is an economically important plant with multiple values as ornamental, for oil production and for medicine. Propagation with conventional methods is limited and tissue culture is a possible alternative. To establish in vitro regeneration system for P. ostii, leaf and petiole were used as explants and plant growth regulators (PGRs) were screened for callus induction, meristematic nodules (MNs) induction, and adventitious shoots formation. The best treatment for callus induction from leaf and petiole was Murashige and Skoog (MS) medium supplemented 1.1 µM thidiazuron (TDZ) + 10.7 µM α-naphthaleneacetic acid (NAA) and 2.3 µM TDZ + 5.4 µM NAA, respectively, with the highest percent of organogenic callus (78.1% and 95.8%, resp.), the highest mean fresh weight (1.5 g and 1.9 g, resp.) and lowest percent of browning (18.2%, and 0.0%, resp.). The best performance of MNs (100.0% and 100.0%, resp.) and leaf clusters induction (42.3% and 43.0%, resp.) from leaf- and petiole-derived organogenic callus were obtained at woody plant medium (WPM) supplemented with 4.0 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) + 2.3 µM TDZ and 2.0 µM CPPU + 4.5 µM TDZ, respectively. The optimum mean number of regenerated adventitious shoots (9.3 and 12.6, resp.) originating from leaf- and petiole-derived callus were obtained at 1.3 µM 6-benzyladenine (BA) + 0.6 µM Gibberellic acid (GA₃), and they achieved the highest rooting (43.3% and 45.5%, resp.) at 4.9 µM indole-3-butyric acid (IBA) treatment.