Monoclonal antibodies against zearalenone (ZEA) were raised in mice according to the hybridoma technology and applied in different immunochemical techniques. More specifically, three formats based on the competitive direct enzyme immunoassay principle were developed: an enzyme-linked immunosorbent assay (ELISA), a flow-through gel-based immunoassay column and a flow-through membrane-based immunoassay. In ELISA, the 50% inhibitory concentration (IC50) was 0.8 ng/mL, and the limit of detection for ZEA standard solutions was 0.1 ng/mL. The antibodies showed a high ZEA (100%) and alpha-zearalenol (alpha-ZOL) (69%) recognition, while cross-reactivities with alpha-zearalanol, zearalanone, beta-zearalenol and beta- zearalanol were 42%, 22%, <1% and <1%, respectively. For standard solutions, a cut-off level at 10 ng/mL could be established for the gel- and membrane-based enzyme immunoassays. Assay time of both non-instrumental tests was 25 min for 10 samples. By including a simple sample extraction procedure, the methods were applied to wheat with IC50s in ELISA of 80 and 120 microg/kg (dilution up to 5% and 15% (v/v) of wheat matrix, respectively). The cut-off level of the gel- and membrane-based immunoassays was established at 100 microg/kg. Potentials and limitations of the developed methods were compared. The possible application for multi-mycotoxin analysis of the ELISA method based on a single monoclonal antibody was investigated. Therefore, principal component analysis and partial least squares regression data modelling were used to separate the immunoassay responses of two cross-reactants (ZEA and alpha-ZOL).