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Background: Ankylosing spondylitis (AS) has been afflicting humankind as far back as ancient Egypt. Macrophage migration inhibitory factor (MIF) appears to have the unique ability to drive both inflammation and new bone formation and could play an important role in the pathogenesis of AS. Objective: The study aimed to evaluate MIF as a diagnostic biomarker in AS. Patients and Methods: This study was conducted on 42 subjects; 21 AS cases and 21 controls. Patients were subjected to full medical history, general and musculoskeletal examinations, laboratory investigations including serum MIF level, sacroiliac X-ray, sacroiliac MRI with the Ankylosing Spondylitis Disease Activity Score (ASDAS) for disease activity. Results: Regarding gender of the patients, the majority (71.4%) of patients were males; while (28.6 %) were females. We found that; the mean disease duration of AS patients was (15.05 ± 6.69) years; while the mean ASDAS score was (2.78 ± 0.68). We also found that, (71.4%) of AS patients received NSAIDs, (95.2%) received diseasemodifying anti-rheumatic drugs (DMARDs), (57.1%) received tumour necrosis factor inhibitors (TNFi). Regarding imaging findings in our study, (61.9 %) of AS patients had sacroiliitis in sacroiliac X-ray while (100 %) of AS patients had sacroiliitis in sacroiliac MRI. Conclusion: Serum MIF levels are elevated in AS patients. ROC-curve analysis showed that the best cutoff point of MIF was (>41.3) with area under curve (0.625), with fair accuracy (57.1%), sensitivity (80%) and specificity (50%).