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A sensitive immunofluorescence assay for rapid AFB1 determination was developed. Polyclonal anti-AFB1 antibody was immobilized onto magnetic nanoparticles (MNPs). The optimal amount of immobilized antibody was found—40 μg antibody per 1 mg MNPs. The conjugate of AFB1 with bovine serum albumin (BSA) and fluorescein isothiocyanate was prepared. UV-Vis and fluorescence spectrophotometric analyses of the obtained conjugate proved the successful conjugate formation. Optimal amount of MNPs with immobilized antibody (0.25 mg) and the optimal concentration of fluorescent conjugate of AFB1 (28 μg/mL) were established. A competitive assay of AFB1 was performed. The toxin solubility and stability were optimal for the assay when using buffer solution with 10% methanol. It was found that the sensitivity of the developed AFB1 assay was significantly higher with 0.1% BSA stabilizing AFB1 solutions. The linear range of the proposed method was 2–100 pg/mL. The immunoassay was applied for determination of AFB1 in spiked peanuts. [ABSTRACT FROM AUTHOR]