A sensitive and specific monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated for the detection of staphylococcal enterotoxin A (SEA). After routine fusion and selection, 10 monoclonal antibodies showed high affinity for SEA. An optimal pair for sandwich ELISA was selected by pairwise interaction analysis. After optimization, the limit of detection (LOD) and linear dynamic range of the method were established, and were found to be 0.0282 ng/mL and 0.06-2 ng/mL, respectively. The recovery in pure milk ranged from 82.67% to 111.95% and the intra- and inter-assay coefficients of variation ranged from 3.16% to 6.05% and from 5.16% to 10.79%, respectively. Cross-reactivity with staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC), staphylococcal enterotoxin D (SED), and staphylococcal enterotoxin E (SEE) in this method were insignificant. These results indicate that the sandwich ELISA method developed in our study is effective for routine identification of SEA in food samples.