A microfluidic paper-based analytical method is developed for determination of Hg(II) using gold nanoparticles functionalized with N,N0 -bis(2-hydroxyethyl)dithiooxamide. Fabrication of the mPAD in this work is free of any expensive equipment and could be performed by untrained personnel. The color of modified AuNPs in the test zone instantly changes after addition of Hg(II), and this color change can be detected by the naked eye, or a digital camera. Under optimized experimental conditions, the linear range is 0.020–8.0 mM of Hg(II) with 12.5 nM (2.5 ppb) detection limit in aqueous solution. In the case of the mPAD, the linear concentration range is 0.025–2.0 mM Hg(II) with a detection limit of 15 nM (3.0 ppb). This method is used for the determination of mercury in real samples such as salmon fish and dusty days in Sanandaj, Iran. The results determined by this method agreed well with those determined according to the ASTM standard method.