The co-administration of several drugs in multidrug therapy may alter the binding of each drug to human serum albumin (HSA) and, thus, their pharmacology effect. Therefore, in this study, the interaction mechanism between HSA and two fluoroquinolones (FQs), sparfloxacin (SPF) and levofloxacin (LVF), was investigated using fluorescence and absorption methods in the absence and presence of the competing drugtigecycline (TGC). The the UV-Vis and fluorescence spectroscopy results showed that the fluorescence quenching of HSA was a result of the formation of the HSA-SPF and HSA-LVF complexes. The fluorescence quenching of HSA-TGC revealed that tigecycline can regulate the binding sites, binding mode and binding affinity of fluoroquinolones. The binding constants (KA) and binding sites (n) of the interaction systems were calculated. The results confirmed that the KA values of the HSA-FQ system decreased in the presence of TGC, indicating that TGC can affect the binding ability of FQ for HSA. This interaction may increase the free plasma concentration of unbound FQ and enhance their pharmacology effect.