Background: Few studies have examined the protein binding ratio of levofloxacin (LVFX) in patients undergoing hemodialysis (HD). Methods: We evaluated the serum concentration and protein binding ratio of LVFX in HD patients. Subjects were 13 patients (7 non-HD and 6 HD patients) undergoing LVFX therapy for pneumonia, urinary tract infection, osteomyelitis, and unidentified infections from June 2012 to December 2013. To clarify the relationship between protein concentration and the protein binding ratio of LVFX in vitro and in vivo, the effect of human albumin and globulin on the binding ratio was investigated. Results: The protein binding ratio of LVFX was 31.2 and 29.3 % in non-HD and HD patients, respectively. A statistically significant correlation was observed between the serum albumin level and protein binding ratio in vivo. LVFX was bound to not only albumin but also globulin; further, in vitro, the protein binding ratio of LVFX increased with an increase in the albumin or globulin concentration. However, the protein binding ratio did not increase with increased serum globulin concentrations in the presence of 3.5 g/dL albumin. Conclusions: The protein binding ratio of LVFX has little clinical relevance, and it is not necessary to monitor it for LVFX therapy in HD patients.