Aflatoxins are highly carcinogenic in nature and occur in a variety of food and feed samples. To ensure food is free from aflatoxins and safe for human consumption, methods to detect these toxins are necessary. Both aflatoxin B 1 and aflatoxin B2 (AFB2) co-occur in a variety of foods. The present study focuses on the generation of single-chain variable antibody fragments (scFvs) that are highly specific to AFB2. A panel of murine scFvs was isolated from a recombinant AFB2 library that was subjected to biopanning against AFB2-bovine serum albumin. The scFvs (E9 and H12) were found to be highly specific to AFB2, with no cross reactivity for other aflatoxins. The analytical potential of these two scFvs was tested by competitive inhibition ELISA and half maximal inhibitory concentrations (IC50) of 85 and 138 ng/ml, respectively, were shown. The affinity and sensitivity of scFv-E9 was determined by kinetic analysis and surface plasmon resonance (SPR)-based competitive inhibition assay using an AFB2-immobilised sensor chip. An SPR-based inhibition assay with IC50 of 8 ng/ml and LOD of 0.9 ng/ml was developed and subsequently successfully applied for AFB2 detection in spiked almond extracts.