Abstract At least three botulinum neurotoxin serotypes, A, B and F, have been implicated in cases of human botulism. The work presented uses a custom-built SPR biosensor to detect each of these three serotypes of neurotoxin in 1 h. Detection curves were established for concentrations of 20 ng/ml and below. The lowest concentration detected for individual toxin samples was either 0.5 or 1 ng/ml for all three serotypes. Detections were also performed on a mixture of all three toxin serotypes in buffer, as well as a 20% honey solution. Detections of mixtures in buffer and in a 20% honey solution complex matrices showed good agreement with detections of individual toxins in buffer.