Our previous report suggested that chitosan nanofiber scaffold may find application in bioartificial livers for it could enhance the function of hepatocytes. This study was focused on its microbiological safety, that is, its influence on the expression and infectivity of porcine endogenous retrovirus (PERV) in primary porcine hepatocytes. Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Exp group and Ctr group) and porcine kidney 15 (PK15) cells were cultured as positive control (defined as PK15 group). The supernatant was removed and substituted with new culture media after 24 hours followed by collecting the media and cells 72 hours later. To quantitative analyze PERV in the supernatant, the PERV RNA and capsid protein gag p30 were detected by dilution technique and RT activity was determined by specific kits. As a result, PERV RNA level, capsid protein and RT activity were all of no significant difference between Exp group and Ctr group, but these indices of the Exp group and Ctr group were both significantly lower than the PK15 group. To determine the expressing level of PERV in the cells, PERV RNA and capsid protein in the cell lysate were detected by RT-PCR and western blot with the software measurement of band lightness. Similarly, there was no difference for the amount of PERV RNA and capsid protein between Exp group and Ctr group but there was a higher PERV RNA level in PK15 group with no significant difference in protein level. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryonic kidney 293 (HEK293) cells, showing no infection. In conclusion, chitosan nanofiber scaffold did not change the low expression and infectivity of PERV in porcine hepatocytes and it was safe for application in bio-artificial livers.