In this paper, we present a microfluidic paper-based analytical device (mu PAD) with a gold nanosensor functionalized with alpha-lipoic acid and thioguanine (Au-TA-TG) to detect whether the arsenic level of groundwater from hand tubewells in Bangladesh is above or below the World Health Organization (WHO) guideline level of 10 g/L. We analyzed the naturally occurring metals present in Bangladesh groundwater and assessed the interference with the gold nanosensor. A method was developed to prevent interference from alkaline metals found in Bangladesh groundwater (Ca, Mg, K and Na) by increasing the pH level on the mu PADs to 12.1. Most of the heavy metals present in the groundwater (Ni, Mn, Cd, Pb, and Fe II) did not interfere with the mu PAD arsenic tests; however, Fe III was found to interfere, which was also prevented by increasing the pH level on the mu PADs to 12.1. The mu PAD arsenic tests were tested with 24 groundwater samples collected from hand tubewells in three different districts in Bangladesh: Shirajganj, Manikganj, and Munshiganj, and the predictions for whether the arsenic levels were above or below the WHO guideline level agreed with the results obtained from laboratory testing. The mu PAD arsenic test is the first paper-based test validated using Bangladesh groundwater samples and capable of detecting whether the arsenic level in groundwater is above or below the WHO guideline level of 10 g/L, which is a step towards enabling the villagers who collect and consume the groundwater to test their own sources and make decisions about where to obtain the safest water.