The study was carried out in selected villages of Jodhpur district of Rajasthan. Study area was divided into four categories with different fluoride levels in drinking water. Total 100 children aged 8-14 years were screened for dental fluorosis and biochemical analysis. 25 children were selected from each category. The dental fluorosis case was determined following dean?s classification. The serum samples of children were investigated to assess FT4, FT3, and TSH hormone levels using Chemiluminiscence Assay by using the Auto analyzer. The level of fluoride naturally was ingested from drinking water and body fluids in all varied over a wide concentration range. The current study also reveals that they have higher-than-normal fluoride content in their body fluids. Serum FT3 levels were recorded highest in category IV 1.91 to 4.42 pg/mL, Mean± SD (3.06±0.78) with minor difference in other groups; concentration of FT4 levels was maximum 0.76 to 1.98 ng/dL, Mean±SD (1.11±0.28) in category III, whereas TSH levels were significantly higher in category IV 0.75 to 4.99 uIU/mL with Mean±SD (1.91±1.10). The children living in endemic fluorosis areas may not have knowledge about thyroid disease and possible association with consumption of drinking water having excess content of fluoride hence they need special care and attention. The results of this study question the validity of the fluoridation of drinking water, food product containing high fluoride to draw attention of public health authorities.