Background and Objectives: Vitamin D deficiency is a significant public health concern worldwide. This study aimed to investigate the relationships between age, sex, and serum vitamin D levels in a cohort of participants from Erbil City. Methods: This cohort study was conducted over a period of two years, from January 2021 to December 2022. A stratified random sampling technique was utilized to guarantee a proportional representation of the population, taking into account both age and sex. The study encompassed a total of 1520 participants ranging in age from 0 to 99 years. The DiaSorin Liaison chemiluminescence assay was utilized to measure serum vitamin D levels. Results:The mean age of participants was 39.461 ± 20.167 years. The overall mean serum Vitamin D level among the study participants was 61.972 ± 32.425 nmol/L. The results indicated that despite an increase in Vitamin D and 25-hydroxyvitamin D levels observed in the second test, a significant proportion of the individuals still exhibited Vitamin D deficiency. Lower Vitamin D levels were particularly noted among younger age groups and females.