Antibody-drug conjugates (ADCs) take the advantage of monoclonal antibodies to selectively deliver highly potent cytotoxic drugs to tumor cells, which have become a powerful measure for cancer treatment in recent years. To develop a more effective therapy for human epidermal growth factor receptor 2 (HER2)-positive cancer, we explored a novel ADCs composed of anti-HER2 scFv-HSA fusion antibodies conjugates with a potent cytotoxic drug DM1. The resulting ADCs, T-SA1-DM1 and T-SA2-DM1 (drug-to-antibody ratio in the range of 3.2-3.5) displayed efficient inhibition in the growth of HER2-positive tumor cell lines and the half-maximal inhibitory concentration on SKBR-3 and SKOV3 cells were both at the nanomolar levels in vitro. In HER2-positive human ovarian cancer xenograft models, T-SA1-DM1 and T-SA2-DM1 also showed remarkable antitumor activity. Importantly, three out of six mice exhibited complete remission without regrowth in the high-dose group of T-SA1-DM1. On the basis of the analysis of luminescence imaging, anti-HER2 scFv-HSA fusion antibodies, especially T-SA1, showed strong and rapid tumor tissue penetrability and distribution compared with trastuzumab. Collectively, the novel type of ADCs is effective and selective targeting to HER2-positive cancer, and may be a promising antitumor drug candidate for further studies.