Botulism is caused by the neurotoxins (BoNTs) produced from Clostridium botulinum. These neurotxins often lead to fatal neuroparalytic disease which is regarded a major threat to the public health. For this reason, rapid and reliable diagnosis of BoNTs in field settings and peripheral care centers is highly valuable. Here, we describe a multiplexed and sensitive optical immunoassay (OIA) for the rapid detection of four medically important BoNTs (A, B, E, and F). The assay is based on detection of physical changes in the thickness of molecular thin film resulting from specific immunobinding events on an optical silicon chip. The immunocomplex causes destructive interference of a particular wavelength of reflected white light from gold to purple-blue on an optical surface depending on the concentration of the analyte. This test allows simultaneous characterization of toxin type and semi-quantitative assessment of toxin level. The assay was four times more sensitive than ELISA when performed with the same reagents. The limit of detection (LOD) for the BoNTs was, respectively, 2.5-5 ng/mL, 5-10 ng/mL, and 10-20 ng/mL in experimentally spiked buffer, water and food matrices. The less logistic load and visual read-out of this method promises potential applicability of this assay in the field as well as in a clinical settings.