The purpose of this study was to develop a method for the extraction and determination of 2,4-dichlorophenoxyacetic acid (2,4-D) from the soil, sediment, and water, and to investigate its stability in water using high-performance liquid chromatography (HPLC). The recoveries of the spiked compound from water and soil samples was found to be in the range of 80 to 100%. The yield of 2,4-D during acidification of the corresponding amine was 93.84-99.98%. A calibration curve for the method showed good linearity (R 2 ≥ 0.9996). The LOD was determined to be 0.45 µg/mL while the LOQ was 2 µg/mL. From the analysis of the samples, 2,4-D was not detected in sediment or soil samples from the Wafiko or Kontola sites (Ethiopia), respectively. The 2,4-D concentrations in soil samples from Bochessa, and water samples from Wafiko and Sher sites (Ethiopia) were high, ranging from 68.22 to 167.7 mg/L which exceeded the United States Environmental Protection Agency (US EPA) regulatory agency standards of 70 µg/L. A 45-day experiment on spiked water samples from Lake Koka (Ethiopia) demonstrated that the acidic form of 2,4-D is stable in water with 73.46 ± 2.00% recoveries. The developed method can be used to determine 2,4-D residues in soil, sediment, and water.