Background High agglomeration of myeloid-derived suppressor cell (MDSC) in neuroblastoma (NB) resulted in immune tolerance and impeded therapeutic effects. Doxorubicin (DOX) is currently found the most specific drug to selectively remove MDSC. However, whether these mechanisms can relieve the inhibitory role of MDSC in NB immunotherapy is still remain unclear. Procedure In this study, the inhibitory role of MDSC for NB Ag-specific cytotoxic T lymphocyte (CTL) were investigated in vitro. CTLs, NB cells, MDSCs and DOX were mixed and cultivated in different collocation pattern, the levels of cluster of differentiation 3ζ chain (CD3ζ) and L-selectin in CTL in different groups were detected. Thereafter, the killing rate of neuroblastoma cells, secretion of interleukin-2 and interferon-γ were detected and compared. Results The proliferation and killing effect of CTLs on neuroblastoma cells were all inhibited by MDSC through downregulating CD3ζ (P = 0.002; P = 0.001) and L-selectin (P = 0.006; P < 0.001) by reai-time PCR test and by Western-blot analysis respectively. However, this inhibitory role can be effectively reversed by doxorubicin. The significant difference existed in the killing rate between the groups (P < 0.001) except between CTL+SK-N-SH group and CTL+SK-N-SH+DOX group (P > 0.05). There were significant differences in the levels of IL-2 (P < 0.001) and IFN-γ (P < 0.001) among the groups. Conclusions This study provided the novel method to enhance immunotherapeutic effects for neuroblastoma by using doxorubicin to targeted inhibition of MDSCs.