Water pollution caused by pesticides is a significant threat to the environment and human health. Silver and gold nanoparticle (AgNPs, AuNPs)-based biosensors are affordable tools, ideal for environmental monitoring. Microfluidic paper-based devices (μPADs) are a promising approach for on-site testing, but few studies have explored the use of laser printing (LP) for μPAD-based biosensors. This study investigates the feasibility of using laser printing to fabricate paper-based biosensors for pesticide detection in water samples. The μPAD was designed and optimized by using different filter paper porosities, patterns, and channel thicknesses. The developed LP-μPAD was used to sense the pesticide atrazine in water through colorimetric assessments using a smartphoneassisted image analysis. The analytical assessment showed a limit of detection (LOD) of 3.5 and 10.9 μM for AgNPs and AuNPs, respectively. The sensor had high repeatability and reproducibility. The LP-μPAD also demonstrated good recovery and functionality in simulated contaminated water. Furthermore, the detection of pesticides was found to be specific under the influence of interferents, such as NaCl and pH levels. By combining laser printing and nanoparticles, the proposed sensor could contribute to developing effective and low-cost solutions for monitoring water quality that are widely accessible.