BACKGROUND: More than 40% patients with diffuse large B cell lymphoma (DLBCL) experienced relapse or refractory (R/R) lymphoma after the standard first R-CHOP therapy. IL-6 was reportedly associated with chemotherapy resistance of rituximab. Further, mesenchymal stem cells (MSCs) are known as the potential cell vehicle for their tropism toward tumor. A MSCs-based tandem diabody for treating DLBCL is currently lacking. METHODS: We constructed a tandem diabody (Tandab(IL-6/CD20)) with modified umbilical cord MSCs (UCMSCs) and designed a cell-based Tandab releasing system. Western blot, qPCR and immunofluorescence were used to confirm the construction and expression of lentivirus-infected UCMSCs. The vitality, apoptosis and homing abilities of UCMSCs were examined via CCK-8 assay, apoptosis, wound healing and migration analysis. Cell binding assay was used to demonstrate the targeting property of Tandab binding to CD20-positive DLBCL cells. Furthermore, we evaluated the viability of SU-DHL-2 and SU-DHL-4 by using CCK-8 and EDU assay after the treatment of UCMSCs-Tandab(IL-6/CD20). RESULTS: Tandab protein peaked at 6273 ± 487 pg/ml in the medium on day 7 after cell culture. The proliferation and homing ability of UCMSCs did not attenuate after genetically modification. Immunofluorescence images indicated the Tandab protein bound to the lymphoma cells. UCMSCs-Tandab(IL-6/CD20) inhibited the growth of SU-DHL-2 or SU-DHL-4 cells in vitro. CONCLUSIONS: UCMSCs-Tandab(IL-6/CD20), which bound with both tumor-associated surface antigens and pro-tumor cytokines in tumor microenvironment, might serve as a potential treatment for DLBCL, evidenced by inhibiting the growth of SU-DHL-2 or SU-DHL-4 cells.