Phosphorus is the limiting nutrient in most freshwater systems, and when found in excess, it can accelerate eutrophication. Eutrophication causes algal blooms, aquatic plant growth, taste, odor problems and oxygen depletion in the water column. Most of the detection techniques are expensive, demand trained person and large reagents and sample volume. The main purpose of this study was to develop inexpensive, simple and fast microfluidic paper- based analytical devices (Î¼PADs) using wax screen-printing technique for the determination of reactive phosphate in water samples. Phosphate is determined by molybdenum blue method using molybdate/antimony reagent and ascorbic acid. Phosphomolybdenum blue complex was formed by adding molybdate/antimony reagent on the hydrophilic circle zone of paper before spotting the sample and reducing agent. The combined reagent was prepared by the ratio of [H+]/ [Mo42-] = 40 - 80 and [Mo42-]/ [Sb] = 21. The produced complex of color intensity was measured by using ImageJ software. Different parameters which are affecting the determination of phosphate were optimized by using Minitab 19.1.2 software of RSM. Under optimal condition, the limits of detection 0.043 mg/L of reactive phosphate of the developed paper device were characterized. The Î¼PAD method showed a very good agreement with UV-Visible spectrophotometry method in recovery study and real sample analysis with paired t-test (0.285) and (0.576), respectively. The RSD of intraday and inter-day were 1.94% and 2.34%, respectively. There was no significant difference between the results obtained using Î¼PADs and UV-spectrophotometer methods paired t-test (0.285). The developed paper-based device is portable, simple, require small volume of reagent and sample, low cost, environmental friendly, suitable for in-situ water quality monitoring and analysis, well-disposed and excellent analytical performance.