A combination of bi-specific antibodies (BsAb), anti-tumor×anti-CD3 and anti-tumor×anti-CD28, is effective in vitro and in vivo, whereas production of two kinds of bi-specific antibodies is labor intensive and administration is complicated. Accordingly, we previously developed a new model of single chain tri-specific antibody (scTsAb), sTRI, which linked both the CD3 and CD28 signals for T-cell activation in one molecule, and demonstrated its capacity for triggering T-cells to kill ovary tumor cells. To improve the pharmacokinetics further and decrease the immunogenicity of scTsAb, we have now generated a new format of scTsAb, TR3H, whose molecular size is smaller than sTRI. Here we describe the construction, purification and characterization of TR3H. TR3H scTsAb bound to effector cells and tumor target cells specifically and induced redirected lyses of ovary tumor cells through freshly isolated, unstimulated human peripheral blood lymphocytes (PBLs). This new format of scTsAb possesses properties that support its potential as a new tumor immunotherapeutic agent.