ABSTRACTBotulinum neurotoxin (BoNT) is the most toxic biomolecule known to the world (⁓100 times toxic than cyanide) and has been listed as category ‘A’ biowarfare agent. In the present investigation, we cloned the catalytic domain of BoNT type ‘F’. Maximum recombinant BoNT/F protein expression was achieved at 21°C, 0.75 mM IPTG at 6 h post induction. The rBoNT/F protein was purified and confirmed subsequently by MS-MS analysis as well as Western blot. High titer polyclonal antibodies were generated and elevated level of IgG1 isotypes was observed. Plate ELISA was developed for the detection of BoNT/F with the sensitivity of 3.9 ng/mL. Limits of Detection (LOD) was established for different fruit juices through spiking studies using rBoNT/F Lc protein as antigen and achieved a detection limit of 15.62 ng/mL for apple juice followed by 31 ng/mL for litchi and 62 ng/mL for grape, orange, and mango juices. The developed system has the potential for the detection of BoNT/F in food as well as environmental samples.