<p>Purpose: Vitamin B12 deficiency is common in India, especially among young adults, often presenting with vague symptoms that delay diagnosis. Early detection through clinical evaluation and lab tests is crucial to prevent serious complications. This study assesses clinical presentations, hematological profile and investigates key laboratory markers aiding accurate diagnosis of B12 deficiency. Methods: A cross-sectional study was conducted on 100 patients attending the Clinical Hematology OPD at tertiary health care centre. Patients with serum B12 levels &lt;300 pg/mL were enrolled. Clinical symptoms, hematological profiles, and laboratory markers including methylmalonic acid (MMA), homocysteine (HC), and holotranscobalamin (HoloTC) were assessed. Results: The majority of patients (61%) were aged 20–40 years, and 69% were male. Anemia was the most common hematological manifestation (58%), followed by bicytopenia (29%) and pancytopenia (13%). Mean Corpuscular volume in severe vitamin B12 deficiency was 111.81 ± 5.09 fL Neurological symptoms were present in 40% of patients, while 62% exhibited neuropsychiatric manifestations, 20 percent patients had gastrointestinal symptoms. Vegetarian diet (34%) followed by alcohol intake (18%) was the most common cause of vitamin B12 deficiency. Holotranscobalmin showed a positive correlation with B12 and was effective in identifying early or subclinical deficiency. Conclusions: Vitamin B12 deficiency presents with diverse clinical and hematological features, even in younger individuals. Incorporating markers like HoloTC and Methyl Malonic Acid, along with clinical assessment, enhances diagnostic accuracy. A stratified, symptom-based diagnostic approach is recommended to improve early detection and treatment outcomes.</p>