Objective To establish an in vivo imaging method of normal or tumorous liver in mice by using a new type nanoparticle contrast agent, ExiTron nano 12000, coupled with micro-CT imaging. Methods Six 6-8-week old male C57BL/6J mice were randomly divided into group A and group group B, by intravenous injection of 50 μL and 100 μL ExiTron nano 12000, respectively. In vivo Micro-CT scans were performed before contrast agent injection, 3 minutes, 24 hours, 7, 14, 28 and 56 days after injection. To determine which dose is suitable for long-term studies, gray scale value analysis was performed on selected region of interest (ROI) in the left lobe and right anterior lobe of the liver, and the changes of liver tissue contrast was monitored after ExiTron nano 12000 injection. Three male HBV transgenic mice bearing liver tumors (group C) were intravenously injected with the determined dose of ExiTron nano 12000 and were monitored by micro-CT scans as above described. At 56 days after ExiTron nano 12000 injection, the mice were sacrificed and liver samples were taken for histological analysis. Results Cross-sectional images taken at various time points and the average gray scale value (AGSV) analysis in the mouse liver revealed that the AGSV peaked at 24 hours after injection of contrast reagent and good contrast still presented in the livers within 56 days of observation for both groups, though group B showed a significantly higher contrast than group A (P<0.01). Those data indicated that the dose of group B (100 μL) was better to maintain ExiTron nano 12000 in the liver of mice for a long time. Contrast-enhanced by 100 μL of ExiTron nano 12000, the liver tumor nodules in the mice of group C could be clearly delineated by Micro CT imaging during a 56 days observation. Histological analysis revealed atypical hyperplasia, enlarged nuclei with hyperchromasia and cell necrosis in the tumors. Conclusions An in vivo imaging method was established to non-invasively visualize mouse liver using micro-CT combined with nanoparticle-based contrast agent and this technology may be applied to a live imaging of murine primary liver tumors.