Mercury is considered the most important heavy-metal pollutant, because of the likelihood of bioaccumulation and toxicity. Monitoring widespread ionic mercury (Hg2+) contamination requires high-throughput and cost-effective methods to screen large numbers of environmental samples. In this study, we developed a simple and sensitive analysis for Hg2+ in environmental aqueous samples by combining a microfluidic immunoassay and solid-phase extraction (SPE). Using a microfluidic platform, an ultrasensitive Hg2+ immunoassay, which yields results within only 10 min and with a lower detection limit (LOD) of 0.13 μg/L, was developed. To allow application of the developed immunoassay to actual environmental aqueous samples, we developed an ion-exchange resin (IER)-based SPE for selective Hg2+ extraction from an ion mixture. When using optimized SPE conditions, followed by the microfluidic immunoassay, the LOD of the assay was 0.83 μg/L, which satisfied the guideline values for drinking water suggested by the United States Environmental Protection Agency (USEPA) (2 μg/L; total mercury), and the World Health Organisation (WHO) (6 μg/L; inorganic mercury). Actual water samples, including tap water, mineral water, and river water, which had been spiked with trace levels of Hg2+, were well-analyzed by SPE, followed by microfluidic Hg2+ immunoassay, and the results agreed with those obtained from reduction vaporizing-atomic adsorption spectroscopy.