We found Listeria monocytogenes was still alive after the ultra-high pressure processing. Based on the result of our former experiments, this research aims to investigate whether the ultra-high pressure processing affects the biofilm formation of L. monocytogenes. 2 strains of L. monocytogenes were applied in the research, both serotypes were 4b.Crystal violet staining assay was used to investigate the biofilm forming capacity of 2 strains, while the fluorescent microscopy and SEM were applied to observe the structures of the biofilm. The biofilm forming capacity of WaX12 was higher than ATCC13932. After 100 MPa UHP processing, WaX12 formed more biofilm than the control group, while 200 MPa improved the biofilm forming capacity of ATCC13932. The biofilm formation of both strains reduced after 300 MPa and higher UHP processing. The images of biofilm formation of the 2 strains obtained by fluorescent microscopy and SEM were consistent with the results of crystal violet staining assay. Results suggested that the UHP processing may increase the biofilm formation of L. monocytogenes. If the pressure was lower than 200 MPa, the biofilm formation was strengthened, and reached the maximum after 48 h. It is important to choose proper pressure during UHP processing.