Background: Acute liver failure (ALF), which can potentially be treated with an artificial liver, is a fatal condition. The purpose of this study was to evaluate the safety and effectiveness of a novel hybrid bioartificial liver system (NHBLS) using simulated liver failure serum in vitro. Methods: The bioreactor in experimental group was cultivated with primary porcine hepatocytes, whereas in control group was not. Next, the simulated liver failure serum was treated using the NHBLS for 10 h. Changes in albumin (ALB), total bilirubin (TBIL), ammonia (Amm), total bile acid (TBA), creatinine (Cr), and blood urea nitrogen (BUN) were measured before treatment (0 h) and every 2 h during treatment. In addition, changes in NHBLS pressures, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lidocaine metabolism were also recorded. Results: The NHBLS worked steadily without unexpected occurrences during the treatment. Blood culture showed no bacterial growth after 7 days, and the endotoxin level was less than 0.5 EU. The TBIL, TBA, Cr, and BUN levels in both groups were markedly lower than those at 0 h ( p < 0.05). The Amm level in experimental group was significantly lower than that in control group ( p < 0.05). NHBLS pressures were also stable, and the hepatocytes in the bioreactor functioned well. Conclusions: The preparation method for the simulated liver failure serum was optimized successfully, and the safety and effectiveness of the NHBLS in vitro were verified. Furthermore, the NHBLS significantly reduced the levels of Amm which can lead to hepatic encephalopathy.