Fusarium mycotoxins such as trichothecenes, zearalenone and fumonisins occur on a worldwide basis in cereal grains, animal feeds and forages. Practical solutions for multiple mycotoxin determination in samples are required by industry and regulators for cost effective screening purposes. The feasibility of developing a novel multiplex nanoarray for the simultaneous and semi-quantitative detection of three regulated mycotoxins: zearalenone (ZEA), T2-toxin (T2) and fumonisin B1 (FUM) was examined. Additionally, the assay was also able to detect HT2 toxin and fumonisin B2 and B3 due to the cross reactivity profiles of the antibodies used. Individual mycotoxin conjugates specific to the three mycotoxins were nano-spotted onto wells of a microtitre plate. Optimisation of assay parameters and antibodies was undertaken with both individual and multiplex calibration curves generated. A competitive assay format was employed enabling a calibration curve for concentration analysis and duplicate results for up to 40 samples in 70 min for the three target mycotoxins. The characteristics and performance of the nanoarray were evaluated including sensitivity and specificity for each target. Additionally, intra and inter spotting precision, cross reactivity, matrix effects and sample analysis in maize and wheat (n=8) was performed. Sensitivity, determined as the concentration causing 50% inhibition, was 70.1, 2.8 and 90.9 ppb in PBS, 172.4, 3.2 and 129.3 ppb in methanol, 197.4, 0.7 and 216.7 ppb in wheat and 43.6, 0.5 and 25.9 ppb in maize for ZEA, T2 and FUM respectively. Intra spotting precision was 6%, 11% and 10% for PBS and 5%, 11% and 12% for methanol for ZEA, T2 and FUM respectively. Inter spotting precision was 4%, 14% and 6% for PBS and 3%, 9% and 16% for methanol for ZEA, T2 and FUM respectively. The feasibility of the nanoarray as an easy to use sensitive screening tool in the 96 well format has been demonstrated for the multiplex detection of three regulated mycotoxins. Improvements in automated image and data analysis software for novice end users are required to improve the overall rapidity of analysis.