Glycolipid chips having a double layer of Au nanoparticles are proposed for detection of biological toxins. The sugar-modified chips constitute an under and an upper layer of Au nanoparticles of 20-80 nm diameter on glass plates, and Au nanoparticles of each layer are linked with 1,8-octanedithiol by a self-assembled monolayer (SAM) technique. A tris-sialo glycosphingolipid, ganglioside GT1b, having lipoic amide at the sphingosine part was immobilized on the Au outside surface of the upper layer, and botulinum toxin (type A heavy chain) was detected by localized surface plasmon resonance (LSPR). The GT1b-Cer-coated chip having a double layer of Au nanoparticles enhanced the toxin detection by LSPR more than those with single monolayers. The LSPR response changed according to the sizes of Au nanoparticles in each under and upper layer. The combination of 60 and 40 nm Au nanoparticles in the under and upper layer, respectively, gave the best result, which enabled the toxin detection at concentrations below 5 ng/mL with the portable LSPR device.