A key challenge for realizing mobile device-based on-the-spot environmental biodetection is that a biosensor integrated with a fluid handling sensor cartridge must have acceptable accuracy comparable to that of conventional standard analytical methods. Furthermore, the user interface must be easy to operate, technologically plausible, and concise. Herein, we introduced an advanced smartphone imaging-based fluorescence microscope designed for Hg2+ monitoring by utilizing a biosensor cartridge that reduced user intervention via time-sequenced passive fluid handling. The cartridge also employed a metal-nanostructured plastic substrate for complementing the fluorescence signal output; this helped the realization of high-accuracy detection, in which a ratiometric dual-wavelength detection method was applied. Using 30 samples of Hg2+-spiked wastewater, we showed that our device, which has a detection limit of ∼1 pM, can perform analytical assays accurately. The detection results from our method were in good linearity and agreement with those of conventional standard methods. We conclude that the integration of a simple-to-use biosensor cartridge, fluorescence signal-enhancing substrate, dual-wavelength detection, and quantitative image data processing on a smartphone has great potential to make any population accessible to small-molecule detection, which has been performed in centralized laboratories for environmental monitoring.