The present study was undertaken to determine the relationship between fluoride in water, urine and serum and dental fluorosis. The fluoride level in water and urine were measured spectrophotometrically by using acid zirconyl and SPADNS reagents, while the fluoride level in serum was determined by ion selective electrode meter. Dental fluorosis survey was conducted with the help of Performa prescribed by Rajiv Gandhi Drinking Water Mission and the use of Tooth Surface Index for Fluorosis. Mean fluoride values in water samples of Jhajjar City and Dadanpur and Dariyapur villages of Jhajjar District were measured to be 2.17 (range from 1.92 to 2.60 mg/L), 2.81 (range from 2.53 to 3.14 mg/L) and 2.22 mg/L (range from 1.63 to 3.33 mg/L), respectively. The mean fluoride values in the urine samples of children were found to be 1.51 (range from 0.05 to 2.64 mg/L), 1.71 (range from 0.69 to 2.80 mg/L) and 1.45 mg/L (range from 0.31 to 2.50 mg/L) at Jhajjar City and Dadanpur and Dariyapur sites, respectively. Serum fluoride was detected in the blood samples of children, who have high urinary fluoride at these three sites. The mean serum fluoride level was reported to be 0.15, 0.34 and 0.17 mg/L, respectively. A total of 842 children were also analyzed for dental fluorosis. The mean values of fluorosis-affected children in Jhajjar, Dadanpur and Dariyapur were 51.90, 94.63 and 36.84 %, respectively. A significantly positive correlation between water, urine, serum fluoride concentration and fluorosis was seen.