Abstract Herbaceous peony (Paeonia lactiflora Pall.) is a landscaping plant that has considerable development prospects worldwide. However, its embryo dysplasia severely limits the breeding of high-quality P. lactiflora cultivars by the cross-breeding method. In this study, the immature natural hybrid embryos of P. lactiflora ‘Zi Fengyu’ with double flower were used to study its rescue and in vitro culture by organogenesis. The results showed that the best medium for adventitious bud induction was modified Murashige and Skoog (MS) medium supplemented with 1.0 mg/L thidiazuron (TDZ), 600 mg/L acidic hydrolysis casein (ACH), 10% coconut water and 100 g/L sucrose. The best medium for adventitious bud multiplication was modified MS medium supplemented with 4.0 mg/L TDZ, 15% coconut water, 60 g/L sucrose and 0.1% activated charcoal (AC). Modified MS medium containing 0.5 mg/L gibberellic acid (GA3), 100 g/L sucrose and 0.1% AC was the more suitable medium for adventitious bud growth. For root regeneration, indole-3-acetic acid (IAA) was a more effective plant growth regulator (PGR), and 1/2 MS medium supplemented with 0.5 mg/L IAA, 30 g/L sucrose and 0.1% AC was the more suitable medium, resulting in a rooting rate of up to 92.12%, and the survival rate was 70.8%. Additionally, histological observation confirmed that the rescue and in vitro culture of P. lactiflora immature embryos by organogenesis was successful. These results provide useful technical support for breeding high-quality P. lactiflora cultivars by the cross-breeding method.