An indirect competitive immunoassay (ic-ELISA) was developed using monoclonal antibody produced by hybridoma AF4, which showed high specificity and reactivity with aflatoxin B1 (AFB1) and aflatoxicol, but low cross-reactivity to other analogs. This low cost reliable method was applied for AFB1 monitoring in the poultry chain of a high agribusiness potential region (northern Paraná state, Brazil). Maize, laying hens feed and egg samples were collected from two poultry farms (with production above 200,000 eggs/day) and evaluated by intralaboratory validated ic-ELISA. The sensitivity of such a validated assay, detecting picogram levels of aflatoxins, demonstrated to be proper for surveying daily ingested cumulative toxins and estimating risks. Additionally, more than 61.00% of positive egg samples ranged between the limit of quantification (LOQ – 0.035 ng/g) and 1.00 ng/g, values commonly not covered by commercial kits. Positive data (>LOQ) occurred in 22 maize (56.40%), 34 feed (85.00%) and 192 (48.00%) egg samples. Mean contamination in maize was 1.51±0.94 ng/g (range 0.11-3.91 ng/g), 1.26±0.96 ng/g in feed (0.10-3.58 ng/g), and 1.01±0.77 ng/g in egg (0.05-3.85 ng/g). No statistical difference was observed between farms (P>0.05) for any of the matrices analysed. However, the difference between median values in maize (0.98 ng/g – Farm A; 1.76 ng/g – Farm B) indicated a higher contamination trend in farm B, possibly due to inadequate local storage. Although there is no limit stipulated for AFB1 contamination in eggs, the levels detected in samples were low and do not represent an immediate risk to animal production or human consumption. Nevertheless, the high frequency of positive maize and feed samples in this field of agribusiness should be highlighted. Sensitive aflatoxin monitoring procedures must be strategically carried out from raw materials to animal derived products, aiming harmless production, which also assures human health.