Objective To accurately and rapidly detect and type five classical Staphylococcal enterotoxins (SEs) by array-ELISA using a combination of a chip and ELISA. Methods SEs were prepared by prokaryotic expression and affinity chromatography. Hybridoma cells were injected intraperitoneally into mice to prepare ascites. A monoclonal antibody was obtained by ascites purification. The sensitivity and specificity of the antibody were evaluated by ELISA. The antibody was printed in one cell, and the sensitivity and specificity of array-ELISA were evaluated. Results Except for the detection limit of Staphylococcal enterotoxin C (SEC) being 10 ng/mL, 0.0001 ng/mL SEs could be detected by array-ELISA in PBS. The detection limit was 0.001-10 ng/mL for SEs in milk. The specificity was 100% in both PBS and milk. No cross reaction was observed between SEs. Additionally, no cross reaction was observed between SEB and botulinum toxin. Conclusion Array-ELISA has been successfully established, and it can simultaneously detect and discriminate five classical SEs within one sample sensitively and specifically.