Background: Trimethylamine N-oxide (TMAO), a gut microbiota metabolite is produced in the liver from dietary precursors such as choline, betaine, and L-carnitine. TMAO has been linked to inflammatory processes and oxidative stress, both of which are critical factors in the progression of hepatitis. This article aims to examine the impact of TMAO on chronic hepatitis B (CHB). Materials and Methods: The study included 41 treatment-naïve CHB patients with HBV DNA levels above 2000 IU/mL, as well as 46 age and gender-matched controls. Serum TMAO levels were measured using Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS). All statistical analysis was performed with R version 4.2.1. Results: Patients with CHB have a more significant increase in serum level of TMAO than healthy controls (1860 [IQR, 808 – 2720] vs. 552.5 [IQR, 252 – 876.5], p 0.001) and had predictive power for CHB with an area under the curve of 0.808. Conclusions: Our results indicate that there is a link between TMAO, a gut microbiota-dependent metabolite, and CHB disease. Since TMAO is synthesized mainly in the liver, its raised levels may be associated with liver-related diseases.