The Hepatitis C virus causes acute and chronic hepatitis. Micro RNA could be used as disease biomarkers. This study looked into Micro RNA125b, Micro RNA 489-3p, and biochemical analysis as a diagnostic for HCV. This study included 20 individuals who had chronic liver disease and were admitted to the Gastroenterology Hospital in Mansoura and the Oncology Institute in Mansoura. All individuals were subjected to full clinical examinations. Following analysis of all patients, polymerase chain reaction (PCR) was confirmed to be positive for chronic HCV in 5 individuals. Five negative control samples were taken from a healthy control group of the same age range and sex. Serum liver function parameters and AFP with gene expression of microRNA125b and microRNA 489-3p were estimated. The HCV group significantly increased AST, ALT, DBIL, TBIL, ALP, GGT, and ALB) while significantly decreasing albumin concentrations. In the HCV group, AFP was substantially higher than in control. The HCV group had a considerable rise in miRNA125b (10.07+ 0.19) but a significant drop in miRNA489-3p (0.36+ 0.02). In addition to biochemical investigation, we found that miRNA125b and miRNA489-3p expression levels were employed as valuable markers for HCV diagnosis.