<p>This cross-sectional study aimed to compare serum vitamin B12, 25-hydroxyvitamin D (25-OHD), ferritin, and iron levels between adults diagnosed with attention deficit hyperactivity disorder (ADHD) and healthy controls, and to evaluate the relationship between these biochemical markers and the severity of ADHD symptoms. In this cross-sectional study, a total of 35 adults with ADHD and 36 age- and sex-matched healthy controls who presented to a psychiatry outpatient clinic in Sakarya in 2024 were included. Participants’ demographic data and clinical characteristics were collected through interviews, while laboratory findings were examined using blood samples taken at the time of inclusion in the study. The severity of ADHD symptoms was assessed using the Adult ADHD Self-Report Scale (ASRS) and the Wender–Utah Rating Scale (WURS). Serum vitamin B12 and 25-hydroxyvitamin D levels were significantly lower in the ADHD group compared with controls (<i>p</i> &lt; 0.001). No significant differences were observed in ferritin and iron levels (<i>p</i> &gt; 0.05). Vitamin B12 and 25-hydroxyvitamin D levels showed moderate-to-strong negative correlations with symptom severity. For example, vitamin B12 levels correlated with ASRS inattention (<i>r</i> = − 0.54, <i>p</i> = 0.00009) and WURS inattention (<i>r</i> = − 0.56, <i>p</i> = 0.000002), while 25-hydroxyvitamin D levels correlated with ASRS hyperactivity/impulsivity (<i>r</i> = − 0.53) and WURS depression (<i>r</i> = − 0.54). These findings indicate that lower serum vitamin B12 and 25-hydroxyvitamin D levels are associated with greater ADHD symptom severity in adults. The results underscore the potential relevance of biochemical factors in adult ADHD and suggest that assessment of vitamin status may be considered in future clinical and research contexts.</p>