We developed a rapid method of total phosphorus (TP) determination in fresh water by optimizing the volumes and concentrations of the oxidant (potassium persulfate), reductant (ascorbic acid), and colorant (molybdate + antimony potassium tartrate). Instead of a UV spectrophotometer, as used in the national standard method (GB 11893-89), we used the iMark Microplate Absorbance Reader to measure the absorbance. The principle of TP determination was identical to that of the national standard method as the water sample was digested under a neutral condition and phosphorus was oxidized to orthophosphate and colored to measure absorbance. In the developed method, we adjusted the volumes of the oxidant and reductant to 80 μL and 0.1 g/mL, respectively, and the concentration of the colorant to 31.00 + 0.32 mol/mL. The water samples collected from Lake Taihu in Maoming, Guangdong, China, were used in the experiment. The feasibility and applicability of the developed method were validated by determining TP in freshwater samples. The results showed that the developed method has excellent accuracy and precision compared with the national standard method. In addition, the national standard method was simplified in terms of the digestion and determination of TP, whereby the volume of chemical reagents and the determination time were reduced. The efficiency of TP determination was improved such that the developed method can be used for monitoring water quality to prevent environmental problems.