Muc1 is one of the most studied tumor antigens. However, antibodies or antibody-toxin conjugates against Muc1 have not shown significant efficacy for tumors with Muc1 overexpression. In this study, we employed bispecific antibody approach to target Muc1 positive tumor cells. A novel bispecific antibody, Muc1-Bi-1, was constructed by linking single domain antibodies, anti-Muc1-VHH and anti-CD16-VHH. Muc1-Bi-2, the humanized form of Muc1-Bi-1, was also constructed by grafting. Both Muc1-Bi bispecific antibodies can be efficiently expressed and purified from bacteria. In vitro, the Muc1-Bi bispecific antibodies can recruit Natural Killer (NK) cells to drive potent and specific cell killing of Muc1-overexpressing tumor cells. In xenograft model, the Muc1-Bi bispecific antibodies can suppress tumor growth in the presence of human peripheral blood mononuclear cells (PBMC). These data suggested that the single domain based Muc1-Bi may provide a valid strategy for targeting tumors with Muc1 overexpression.