Background: Repeated blood transfusion remains a cornerstone in the management of chronic hematological disorders but is associated with significant biochemical and hematological alterations, including iron overload, hepatic dysfunction, and electrolyte imbalance. Objective: To assess biochemical and hematological changes associated with repeated blood transfusion among patients attending a tertiary hospital. Methods: This hospital-based comparative cross-sectional study involved 120 participants comprising 80 patients who had received ≥3 blood transfusions in the preceding 12 months and 40 age- and sex-matched controls with ≤1 transfusion. Hematological parameters were analyzed using an automated hematology analyzer, while biochemical assays were performed using standard spectrophotometric methods. Data were analyzed using SPSS version 25. Independent t-test, Pearson correlation, and linear regression were applied. A p-value <0.05 was considered statistically significant. Results: Repeatedly transfused patients had significantly higher mean serum ferritin (486.3 ± 162.5 ng/mL vs 168.4 ± 72.1 ng/mL; p<0.001), ALT, AST, bilirubin, and potassium levels compared to controls. Serum calcium was significantly lower in transfused patients (p=0.02). Serum ferritin showed a strong positive correlation with transfusion frequency (r=0.64, p<0.001). Conclusion: Repeated blood transfusion is associated with significant biochemical and hematological alterations, particularly iron overload and hepatic dysfunction. Routine laboratory monitoring is recommended for early detection and prevention of transfusion-related complications.