In the present study, we introduce a novel hybrid sandwich-ALISA employing chicken IgY and ssDNA aptamers for the detection of staphylococcal enterotoxin B (SEB). Cloning, expression and purification of the full length recombinant SEB was carried out. Anti-SEB IgY antibodies generated by immunizing white leg-horn chickens with purified recombinant SEB protein and were purified from the immunized egg yolk. Simultaneously, ssDNA aptamers specific to the toxin were prepared by SELEX method on microtiter well plates. The sensitivity levels of both probe molecules i.e., IgY and ssDNA aptamers were evaluated. We observed that the aptamer at 250 ngmL(-1) concentration could detect the target antigen at 50 ngmL(-1) and the IgY antibodies at 250 ngmL(-1), could able to detect 100 ngmL(-1) antigen. We further combined both the probes to prepare a hybrid sandwich aptamer linked immune sorbent assay (ALISA) wherein the IgY as capturing molecule and biotinylated aptamer as revealing probe. Limit of detection (LOD) for the developed method was determined as 50 ngmL(-1). Further, developed method was evaluated with artificially SEB spiked milk and natural samples and obtained results were validated with PCR. In conclusion, developed ALISA method may provide cost-effective and robust detection of SEB from food and environmental samples.