We synthesized a new botulinum neurotoxin type A (BoNT/A) sensing material consisting of a photopolymerizable polyacrylamide hydrogel incorporating a 25-mer BoNT/A recognizable peptide substrate coupled to an acryloyl-PEG-NHS crosslinker that readily reacts with free amines at pH 8-9 via the NHS moiety. The peptide substrate residues are slightly modified from synaptosomal-associated protein SNAP-25 between amino acid residues 187-203 with an additional three glycine spacers and a lysine on each end for coupling to the crosslinker via NHS-ester conjugation reactions. The alpha and epsilon amines of the N-terminal lysine and epsilon amine of the C-terminal lysine were used to couple the peptide to the crosslinker instead of previously used cysteines to avoid having to remove dithiothreitol (DTT) after sample preparation or potential false positive substrate cleavage by DTT if not removed after sample preparation. By integrating this hydrogel into a microchannel, we developed a BoNT sensor based on the morphology change of this hydrogel. The hydrogel in the microchannel can be degraded in 22 h at 45 ?g/mL of light chain (LC) of BoNT/A and 90 h at the lowest concentration of 4.5 ?g/mL of LC, but remains intact in Hepes buffer and DTT solutions. The results indicate that the hydrogel-based device can potentially be a portable sensor for the detection of active BoNT/A with high accuracy and specificity.