The aim of the present study was to construct a single-chain bispecific antibody (scBsAb) against cervical carcinoma and to investigate its biological activities. The scBsAb was constructed using a genetic cloning technique and antigen binding activities were detected by ELISA. The iodogen method was used to analyze the pharmacokinetics. The Rosette formation test was used to detect the binding ability between peripheral blood lymphocytes (PBLs) and Cs1213 cervical cancer cells. In addition, the MTT method was performed to detect the killing effect of PBLs. The molecular weight of the scBsAb was ~60 kDa. The antigen binding activities of scBsAbs were compared with the anti‑human cervical carcinoma antibody single‑chain Fv fragment (CSAs‑1 scFv) and anti‑cluster of differentiation (CD)3 scFv (P>0.05). In addition, a pharmacokinetics assay demonstrated that compared with the two corresponding scFvs, scBsAbs exhibited a significantly prolonged retention time in the body (P<0.01). In addition, the number of rosettes formed by PBLs and Cs1213 cells in the scBsAb group was markedly greater than that in the scFv groups or the RPMI‑1640 group (P<0.05 and P<0.01, respectively). The killing activity of PBLs against scBsAb‑mediated Cs1213 cells was significantly greater than that mediated by the other antibodies (P<0.05). When the concentration of scBsAb was 40 µg/ml, the killing rate was 64.5%. Thus, anti‑human cervical carcinoma/anti‑CD3 scBsAbs may possess two types of antigen binding activity, prolong the duration in vivo and improve the killing activity of PBLs against cancer cells.