A simple and sensitive fluorescence immunoassay for the detection of aflatoxin B(1) (AFB(1), as a model compound) in food was developed using AFB(1)-bovine serum albumin conjugate (AFB(1)-BSA)-functionalized magnetic beads as immunosensing probes. The recognition elements were prepared by doping of rhodamine B (RB) fluorophore into silica nanoparticles followed by immobilization of monoclonal anti-AFB(1) antibodies on the silica shell. Based on a competitive-type immunoassay format, the assay was performed both in low-binding polypropylene 96-well microtiter plates (MTPs) and in an automated sequential injection (SI) format. Similar detection limit (LOD) of 0.2 ng mL(-1)vs. 0.1 ng mL(-1) but narrower dynamic working linear range of 0.5-7 ng mL(-1)vs. 0.5-30 ng mL(-1) was obtained toward AFB(1) standards with the flow setup compared to the MTP format. Intra-batch assay precision was substantially improved (≤5.3% vs.≤8.7%) by resorting to the SI manifold. The proposed method features unbiased identification of negative (blank) and positive samples. No significant differences at the 95% confidence level were encountered in the analysis of naturally contaminated peanut samples between the proposed immunoassay and liquid chromatography for determination of AFB(1).