This paper reported a real-time detection strategy for Hg²⁺ inspired by the visible spectrophotometer that used a smartphone as a low-cost micro-spectrometer. In combination with the smartphone’s camera and optical accessories, the phone’s built-in software can process the received light band image and then read out the spectral data in real time. The sensor was also used to detect gold nanoparticles with an LOD of 0.14 μM, which are widely used in colorimetric biosensors. Ultimately, a gold nanoparticles-glutathione (AuNPs-GSH) conjugate was used as a probe to detect Hg²⁺ in water with an LOD of 1.2 nM and was applied successfully to natural mineral water, pure water, tap water, and river water samples.