A simple, precise, accurate, rapid, economical and sensitive Uv spectrophotometric method has been developed for the determination of Chloramphenicol in pharmaceutical preparations and environmental wastewater samples, which shows maximum absorbance at 281 nm in a solution of methanol: H2O (10 in 1000) (v/v) was used as a solvent. Beer's law was obeyed in the range of 0.05 - 0.6 mg/ ml, with molar absorptivity of 6.6×105 L.mol-1.cm-1, relative standard deviation of the method was less than 1.6%, and accuracy (average recovery %) was 100 ± 1.0. The method was successfully applied to the determination of Chloramphenicol in some pharmaceutical formulations (Eye drops 0.5% and Eye Ointment1%) and industrial wastewater samples. The proposed method was validated by sensitivity and precision which proves suitability for the routine analysis of Chloramphenicol in true samples.