Objectives: This present study was to evaluate the association of various parameters like TSH levels, FT4, FT3 and anti TPO levels with the levels of vitamin D3 in hypothyroidism patients. Methods: Detail history clinical examinations and relevant investigations were performed to all subjects. Sample was collected for the estimation of serum TSH, FT3, FT4, Anti–TPO and Vitamin D3 levels. Under complete aseptic conditions venous blood was withdrawn from antecubital vein. Levels of FT3, FT4, TSH, Anti–TPOAb and Vitamin D3 were estimated using fluorescence array. Results: Data was analyzed by using SPSS version 26 software. One sample statistical methods analysis was used. Mean ± standard deviation were calculated. P value was taken less than or equal to 0.05 for significant differences (p ≤ 0.05). Conclusions: Hypothyroidism patients had significantly increased TSH levels. Most of the patients had decreased vitamin D3 levels. And the patients who had increased TSH levels were associated with decreased vitamin D3 levels. Hence, all the patients of hypothyroidism should be assessed for the levels of vitamin D3. And accordingly vitamin D3 supplementation should be advised for early management and treatment of vitamin D3 deficiency in hypothyroidism patients.