Organophosphorus (OPs) pesticide residues pose significant threats to human health and the environment. To tackle this issue, we synthesized water-soluble fluorescent conjugated polymer nanoparticles (WSCPNs), which offer high fluorescence intensity, simple preparation methods, and ease of functionalization, making them ideal candidates for fluorescent sensing applications. These WSCPNs were subsequently used to prepare a WSCPNs@MnO2 probe via in situ synthesis, resulting in efficient fluorescence resonance energy transfer between WSCPNs and MnO₂. This system effectively oxidizes non-fluorescent o-Phenylenediamine (OPD) into 2,3-diaminophenazine (DAP). In the absence of OPs, acetylthiocholine (ATCh) is catalyzed by acetylcholinesterase (AChE) to produce thiocholine (TCh), which reduces MnO₂ on the surface of the probe, restoring the fluorescence intensity. When OPs are present, AChE's catalytic pathway is inhibited, limiting the recovery of fluorescence intensity in WSCPNs. The remaining MnO₂ can further oxidize OPD to DAP, allowing quantitative analysis by monitoring changes in fluorescence signal ratios, achieving a detection limit of 0.0139 ng/mL. Additionally, color changes can be captured and analyzed using a smartphone, facilitating fluorescence visualization for OPs detection, achieving a detection limit of 0.025 ng/mL. This method exhibits excellent anti-interference capabilities and has been successfully applied to detect organophosphorus pesticides in leaves and soil, demonstrating the effectiveness of our ratiometric fluorescence and fluorescence visualization dual-mode sensing platform for monitoring OPs.Copyright © 2024 Elsevier B.V. All rights reserved.