The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) is a major target in the development of diagnostic assays and vaccines, but its antigenic and immunogenic properties remain unclear. Seven SARS-CoV spike proteins (S, SQ, S1, RBD, S2, S2Q, and CX) were generated using the modified vaccinia virus (Tiantan strain) as a vector, and their antigenicity and immunogenicity were evaluated. The secreted SQ protein in which the transmembrane domain was deleted, as well as the full-length spike protein, showed the most potential to induce the production of neutralizing antibody (nAb) in mice. S1 and RBD proteins initialized significantly lower levels of nAb production. In addition, the S proteins were recognized specifically by the sera of convalescent patients with SARS, and that of mice immunized with inactivated SARS-CoV, but did not react with anti-sera of HCoV-OC43 or HCoV-229E, or sera from healthy donors (although RBD showed a false-positive in 1 of 55 control samples of human sera). Our results demonstrate that SQ protein may be an effective vaccine candidate and a convenient and safe diagnostic antigen for SARS-CoV.