Objective To observe and evaluate the short-term therapeutic effect of intravitreal injection with topotecan for refractory vitreous seeding from retinoblastoma (RB)． Methods Eleven patients (11 eyes) of RB with refractory vitreous seeding (received intravenous chemotherapy, intra-arterial chemotherapy, intravitreal melphalan, laser, cryotherapy and subsequently developed refractory viable vitreous seeds) were enrolled in this study. There were 6 males (6 eyes) and 5 females (5 eyes). The aged from 9 to 44 months, with the mean age of 26 months. According to International Intraocular Retinoblastoma Classification, 11 eyes were initially classified as group E (3 eyes), D (6 eyes), B (1 eye) or A (1 eye). All patients were received intravitreal injection with topotecan. A total of 32 intravitreal topotecan injections were performed with a mean of 2.9 injections (median 3 injections; range 2−4 injections). The mean follow-up was 10 months. The safety and effectiveness of intravitreal injection with topotecan for refractory vitreous seeding from RB were observed. Results Complete regression of vitreous seeds was achieved in 11 of 11 eyes (100%), including complete disappearance in 9 eyes and fibrosis in 2 eyes. None of the patients needed enucleation and occured ocular or systemic complications in the follow-up period. Conclusion Intravitreal injection with topotecan for refractory vitreous seeds from RB is effective and safe.