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The present study investigates the effects of input wavelength (1064, 532, and 355 nm) and surrounding liquid environment (distilled water and aqueous NaCl solution) on the picosecond laser ablation on silver (Ag), gold (Au), and Ag/Au alloy targets. The efficacy of the laser ablation technique was meticulously evaluated by analyzing the ablation rates, surface plasmon resonance peak positions, and particle size distributions of the obtained colloids. The nanoparticles (NPs) were characterized using the techniques of UV-visible absorption, transmission electron microscopy, and energy-dispersive X-ray spectroscopy. Furthermore, NPs of various sizes ranging from 6 to 35 nm were loaded onto a filter paper by a simple and effective drop-casting approach to achieve flexible surface-enhanced Raman spectroscopy (SERS) substrates/sensors. These substrates were tested using a simple, portable Raman device to identify various hazardous chemicals (malachite green, methyl salicylate, and thiram). The stability of the substrates was also systematically investigated by determining the decay percentages in the SERS signals over 60 days. The optimized SERS substrate was subsequently employed to detect chemical warfare agent (CWA) simulants such as methyl salicylate (a CWA simulant for sulfur mustard) and dimethyl methyl phosphonate (has some structural similarities to the G-series nerve agents) at different laser excitations (325, 532, and 633 nm). A notably higher SERS efficiency for CWA simulants was observed at a 325 nm Raman excitation. Our findings reveal that a higher ablation yield was observed at IR irradiation than those obtained at the other wavelengths. A size decrease of the NPs was noticed by changing the liquid environment to an electrolyte. These findings have significant implications for developing more efficient and stable SERS substrates for chemical detection applications., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024, Moram et al.)