In order to detect deoxynivalenol (DON) contamination in cereals, a time-resolved fluoroimmunoassay (TRFIA) was developed. By use of DON-bovine serum albumin (DON-BSA), anti-DON antibody, and europium-labeled goat anti-rabbit antibody, this study established an indirect competitive DON-TRFIA. The sensitivity of this method was high, with the detection limit of 0.01 ng/mL. The tests showed the sensitivity was 0.1 microg/kg in cereals and 0.1 ng/mL in beer. Its detection range was 0.01-100 ng/mL. Within the detection range, the intra- and inter-batch CVs of the DON-TRFIA were 3.6% and 8.2%. Data obtained from corn and beer samples by TRFIA and ELISA were in good agreement. This assay did not cross-react with zearalenone (ZEN) and nivalenol (NIV) which commonly exists in corn. This study suggests that DON-TRFIA is a simple, fast and economic method for screening large quantities of samples, and has good prospects of application.