Trem-like transcript 2 (TLT-2), one of the TREM family members, which is expressed on B cells, T cells, and macrophages, plays a critical role in immune response mechanism. In this study, two novel mouse anti-human TLT-2 monoclonal antibodies (MAbs) were prepared using hybridoma technology and their immunological characteristics were determined. The results showed that the two MAbs (clones 10F5 and 8C10) were both IgG1 (κ) and bound specifically to human TLT-2. Furthermore, 10F5 and 8C10 seemed to recognize a different site (epitope) of TLT-2 by competition assay. MAb 10F5 was proven in Western blot analysis to specifically bind to denatured TLT-2 protein while both MAbs were proven in dot blot analyses and immunofluorescence to specifically bind to natural TLT-2 protein. In addition, crosslinking of TLT-2 with MAb 8C10 markedly blocked TLT-2 positive signal and T cell proliferation. Taken together, these two monoclonal antibodies might be of great value as tools for further exploration of the expression and function of TLT-2.