An ultrasensitive fluorimetric biosensor for the detection of chemical warfare agent sulfur mustard (SM) was developed using its monofunctional analogue. SM is a vesicant and a potent chemical threat owing to its direct toxic effects on eyes, lungs, skin and DNA. This work investigates the quenching action of the analyte on chlorophyll fluorescence as elucidated by nuclear magnetic resonance, Fourier transform infrared spectroscopy and mass spectrometry studies suggesting the electrophilic attack of carbonium ion on nitrogens of the porphyrin moiety of chlorophyll. The properties of immobilisation matrix were optimised and scanning electron microscope observations confirmed improvement in pore size of sol-gels by addition of 32 % (v/v) glycerol, a feature enabling enhanced sensitivity towards the analyte. Chlorophyll embedded sol-gel was treated with increasing concentrations of monofunctional SM and the corresponding drop in maximum fluorescence intensity as measured by emission at 673 nm was observed, which varied linearly and had a detection limit of 7.68 x 10(-16) M. The biosensor was found to be 6 orders of magnitude more sensitive than the glass microfibre-based disc biosensor previously reported by us.