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.