A fluoroimmunoassay towards aflatoxin B1 (AFB1) was presented using quantum dots as the fluorescent label. The CdTe QDs were successfully linked to the monoclonal antibody against AFB1. Based on the conjugated complexes, a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) was developed for AFB1 detection. The 50% inhibition value (IC50) of the cFLISA was 0.149 ng/mL in peanuts matrix. The method performance included the limit of detection (LOD) of 0.016 ng/mL and considerable recoveries of 85–117% at three fortification levels (0.075, 0.15, and 0.3 ng/g) from spiked AFB1 blank peanuts samples, along with coefficients of variation (CVs) below 10%. The cFLISA provided an alternative of rapid and sensitive detection for AFB1 and, moreover provided great potential for multiplexed mycotoxins determination simultaneously.