An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody was established to detect aflatoxin B1 (AFB1) in tea. The antibody was prepared from a hybridoma derived by fusing Sp2/0-Ag14 myeloma cells and immunised spleen B cells. The effects from pH, ionic strength, and organic solvents on immunoassay were optimised and the 50% inhibition (IC50) value was 0.057 ± 0.007 ng mL−1. Spiked black and green tea samples at 10, 20 and 50 ng g−1 levels of AFB1 were detected with this proposed ELISA. The recoveries for black tea samples ranged from 68.5% to 117.7% and 73.5 to 114.3% for green tea samples. This immunoassay showed no cross-reactions with other mycotoxin family but good recognition with related aflatoxins. These results indicate that the ELISA assay could be used as a screening method for aflatoxin detection in tea samples.