Background Cancer bladder is the most common malignant tumor affecting the urinary tract. Genetic alterations are tightly associated with the development of cancer bladder. MicroRNAs (miRNA) are small, noncoding single-stranded RNA mol- ecules that have been linked to bladder cancer. miR-124-3pa exhibits altered expression in various types of human malignan- cies. DNA methyltransferase 3B (DNMT3B) is responsible for de novo DNA methylation which is a fundamental epigenetic process in carcinogenesis. This work was performed to study the expression of DNMT3B and miR 124-3pa in bladder cancer tissues, and investigate their significance in the diagnosis and prognosis of the disease. Subjects & methods This case-control study included one hundred and six tissue samples of patients with primary urothe- lial bladder cancer. The tissues were separated into two parts. The first part was immediately frozen and kept at −80 °C for total RNA extraction with subsequent detection of miR 124-3pa and DNMT3B expressions. The other part was preserved in formalin solution for histopathological examination. Results There was a highly statistically significant difference between the cancerous and the normal tissues as regarding miRNA-124-3pa and DNMT3B expression (P <0.001) for each. Also, there was a highly statistically significant strong nega- tive correlation between miRNA-124-3pa and DNMT3B expression (r=-0.750, P <0.001). The combined performance of miR-124-3pa and DNMT3B revealed that the cutoff point of ≥3.3 can be used as a predictor of the presence of cancer bladder with sensitivity of 98.1% and specificity of 80%. Conclusion miR-124-3pa and DNMT3B can be used as predictors of the presence of cancer bladder.