This study explores the colorimetric sensing of chlorpyrifos (CP) pesticides using neem root extracts-synthesized silver nanoparticles (NRE-AgNPs). The NRE-AgNPs were synthesized via a cost-effective bio-reduction method from neem (Azadirachta indica) and evaluated for their pesticide’s  colorimetric sensing property. Phytochemical analysis confirmed the presence of tannins (80.4 mg/mL), phenols (60.8 mg/mL), flavonoids (54.6 mg/mL), alkaloids (28.9 mg/mL), reducing sugars (59.1 mg/mL), and cardiac glycosides (47.6 mg/mL), which facilitated nanoparticle formation and stability. Scanning electron microscopy revealed nanoparticles with an average particle size of 68.3 nm, while energy-dispersive X-ray spectrum confirmed the presence of silver (0.14% atomic, 0.25% weight) alongside stabilizing organic compounds. UV-Vis spectroscopy confirmed NRE-AgNP synthesis with a surface plasmon resonance peak at 405 nm. The nanoparticles responded sensitively to the presence of the chlorpyrifos, especially at 300 to 350 nm, marking a clear deviation from their original 405 nm signature, thus confirming the viability of AgNPs as an optical sensor. This study underscores the potential of neem root-based AgNPs as a sustainable and eco-friendly solution for pesticide sensing, offering promising applications in the environmental field.