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The pre-aflatoxin protein biomarker PAB-01 is a key target for early predicting of aflatoxin occurrence risk. Improving its detection sensitivity is significant for the early evaluation of potential risks. In this study, we obtained the monoclonal antibody 6A6 against PAB-01 through hybridoma technology. A sandwich ELISA was developed using pairwise combinations of PAB-01 nanobody, monoclonal antibody, and polyclonal antibody. It was found that the nanobody as the capture antibody and monoclonal antibody as the detection antibody exhibited the highest sensitivity, with a detection limit of 0.92 ng/mL. Based on this, a rolling circle amplification-based immunoassay (RCA-EIA) was established, with a linear range of 0.056–41.152 ng/mL and a detection limit of 0.033 ng/mL. Recovery rates in spiked peanut and corn samples ranged from 94.49% to 127.50%. This method successfully identified the presence of the PAB-01 protein in maize samples 10 days prior to the aflatoxin production. Based on PAB-01 concentration classifications, the maize samples were categorized into different risk levels. This development is significant for early warning and risk stratification management of aflatoxin occurrence, providing a highly effective tool in ensuring food safety. • A new monoclonal antibody against PAB-01 protein was obtained. • VHH capture and mAb detection yielded the highest sensitivity in sandwich ELISA. • A rolling circle amplification (RCA)-based sandwich ELISA method was established. • RCA-EIA detected PAB-01 giving a 10-day early aflatoxin warning in corn. • Potential risk of aflatoxin occurrence was classified based on PAB-01 concentration. [ABSTRACT FROM AUTHOR]