Background: The most prevalent kind of spondylo-arthropathies is ankylosing spondylitis (AS); >90% of those who have it are HLA-B27 positive. AS may be influenced by non-HLA genes. MicroRNAs are 18-23 nucleotide non-coding RNA molecules that gene expression control post-transcriptional. Objective: The aim of the present study is to evaluate the expression profile of miRNA-125b and serum levels of ESR, CRP in the control group and patients alike and find their association with disease progression in AS patients and evaluate their significance as novel markers for AS. Patients and methods: This case control study included 100 AS male patients and 100 healthy controls of matched age with no history of infectious or autoimmune disorders. AS patients were recruited from the Hematology Clinic in Medical City (Baghdad Hospital) and diagnosed with AS on the basis of complete blood picture (CBC), CRP, along with MRI, and X-Ray. Clinical assessment, history of treatment administration, disease duration, and smoking were taken into account for Disease Activity Indexes for AS patients. Disease was assessed using the Bath AS Disease Activity Indicator (BASDAI), along with ESR (mm/h) and (CRP) (mg/l) to assess the mobility and functional limitations. We investigate (miRNA-125b) expression in individuals with AS and in healthy individuals by real-time quantitative polymerase chain reaction. Characteristic Receiver operating (ROC) in AS patients was done to evaluate accuracy of diagnostic of miRNA-125b in patients with AS from control subjects. Results: AS male patients had mean age of 40.56 (SD 10.19) and disease duration of 9.04 (SD 2.03) years. The age of the male controls (38.78 ± 10.57 years) was not significantly different from that of AS patients (p=0.227). Both AS Disease Activity Index (BASDAI) and AS Functional Index (BASFI) values were 3.45 (SD 2.03) and 3.84 (SD 2.62), respectively. It was observed that 57% of patients have HLA-B27positive and ESR and levels C-reactive protein were 18.34 (SD 3.45) and 25.39 (SD 19.59) mm/h, respectively. Our results revealed a significant decrease in (miRNA-125b) expression with fold change (0.133) in AS patients. The area under curve (AUC) was 0.962 and cut off point was 8.94. Specificity and sensitivity of miRNA-125b were 92% and 91%, respectively. Conclusion: Expression profile of miRNA-125b can be used as novel markers for AS.