The aim of the present study was to evaluate the diagnostic relevance of urinary fibronectin (FN), telomerase (RTA), and cytokeratin 20 (CK20) mRNA in comparison with voided urine cytology (VUC). The study included 132 patients with bladder cancer, 60 patients with benign bladder lesions, and 48 healthy individuals. All were subjected to urine cytology, estimation of fibronectin by ELISA, RTA by TRAP, and CK20 mRNA by conventional RT-PCR in urothelial cells from voided urine. The best cutoff point for FN was determined by receiver operating characteristic curve (41.7 ng/mg protein) revealed the highest sensitivity for malignant (80%) followed by the benign (70%) than the healthy individuals (4.1%) at P < 0.001. Also, RTA and VUC showed significant difference among the three investigated groups (P < 0.001). The overall sensitivity (89.3%) and specificity (98.4%) were the highest for CK20 mRNA. Combined sensitivity of VUC with FN, RTA, and CK20 mRNA together (98.4%) was higher than either the combined sensitivity of VUC with any of them or than that of the biomarker alone. Accordingly, when the diagnostic efficacy was considered, CK20 mRNA had the highest sensitivity and specificity compared to all investigated markers.