The binding of ciprofloxacin (CIP) to human holo-transferrin (HTF) in the presence of silver nanoparticles (AgNPs) has been investigated by fluorescence quenching and circular dichroism (CD) techniques as well as resonance light scattering under physiological conditions. It was determined that the intrinsic fluorescence of HTF was quenched by CIP in the presence of AgNPs through static quenching, thus confirming that a CIP-HTF complex was formed in both the binary and ternary systems. However, the analysis of HTF fluorescence quenching in these binary and ternary systems indicated that the AgNPs were affected upon complex formation between CIP and HTF and that the binding affinity between them became more substantial when the AgNPs coexisted with the drug. Fluorescence quenching proved that HTF had one class of binding sites for CIP in both binary and ternary systems. CD spectra indicated that the secondary structure of HTF changed when increasing the CIP concentration and during the simultaneous presence of CIP and AgNPs, which led to decreased contents of