The aim of this study is to explore the effect of flavonoids from Rosa roxburghii Tratt (FRRT) on doxorubicin (DOX)-induced autophagy of myocardial cells. Primary isolation and culture of myocardial cells and H9C2 myocardial cell lines from 1 to 3-day-old rats were performed, myocardial cells were incubated using 5 μmol/L DOX and a cardiotoxicity model was established, intervention was conducted via FRRT, and the ultrastructure of myocardial cells was observed under a transmission electron microscope. The expressions of LC3-II and P62 proteins were detected through immunofluorescence and Western blotting. The ultrastructure showed a large quantity of autophagic vacuoles of the cells in DOX group with poor cell state. After the FRRT intervention, only a small quantity of autophagic vacuoles appeared in the myocardial cells, and there were many coarse microvilli on the cell surface. The expression of P62 protein was reduced in DOX group, while that in FRRT group was increased (p < 0.01). In conclusion, FRRT exerts a protective effect in the DOX-induced cardiotoxicity by down-regulating DOX-induced autophagy of myocardial cells.