We herein report a novel fluorimetric detection method for Hg²⁺ and Pb²⁺ based on in situ formation of AgNPs, where thiocholine (TCh), a product obtained by the hydrolysis of acetylcholine (ACh) by acetylcholinesterase (AChE), can act as a stabilizing agent. Uniformly dispersed AgNPs were obtained in the control sample (in the presence of optimized concentrations of AChE, ATCh, AgNO₃ and NaBH₄) without addition of any inhibitors (Hg²⁺ or Pb²⁺), resulting in decreased fluorescence intensity. However, the addition of Hg²⁺ and Pb²⁺ to the reaction mixture tends to decrease the vibrational and rotational speed with an increase in the aggregate diameter of particles, and thus increases the emission intensity of AgNPs at 423 nm. The increase in the emission intensity can be well correlated to the increase in concentration of metal ions. The limit of detection (LOD) was found to be 1.47 × 10⁻¹⁴ M for Hg²⁺ and 0.324 × 10⁻¹⁰ M for Pb²⁺, and the proposed method was successfully used for the determination of Hg²⁺ and Pb²⁺ in environmental samples. Further, this sensor model was also employed to perform the concurrent detection of Hg²⁺ in the presence of Pb²⁺ or vice versa in aqueous solutions.