The interactions between cyclophosphamide (CYC) and lysozyme (LYZ) in the presence of different cyclodextrins (CDs) were investigated by UV absorption, fluorescence spectroscopy, circular dichroism (CD), and molecular modeling techniques under imitated physiological conditions. The UV absorption results showed the formation of complexes between CYC and LYZ in the presence of different CDs. Fluorescence data show that CYC has a stronger quenching effect on LYZ, and the red shifts suggested that the microenvironment of Trp residues was changed and became more hydrophilic. The interaction of CYC with LYZ and quenching properties of the complexes caused strong static fluorescence quenching in binary and ternary systems. The binding affinities as well as the number of binding sites were obtained from interaction between CYC and LYZ in the presence of different CDs as binary and ternary systems by modified Stern-Volmer plots. The Resonance Light Scattering (RLS) technique was utilized to investigate the effect of drug and CDs on conformational changes of LYZ as separate and simultaneous. The results suggested that the enhancement of RLS intensity was attributed to the formation of a complex between drug and protein in absence and presence of CDs. The effect of CYC and cyclodextrins on the conformation of LYZ was analyzed using synchronous fluorescence spectroscopy. Our results revealed that the fluorescence quenching of LYZ originated from the Trp and Tyr residues, and demonstrated conformational changes of LYZ with the addition of CYC and CDs. The molecular distances between the donor (LYZ) and acceptor (CYC and CDs) in binary and ternary systems were estimated according to Forster's theory and showed static quenching for protein with CYC in the presence of CDs. The CD spectra indicated that the binding of the CYC induced secondary structural changes in LYZ in binary and ternary systems. Molecular modeling suggested the binding sites of CYC in the ternary systems differ from those in the binary systems. estimated the distance between CYC and Trp residues in binary and ternary systems in the presence of CDs and confirmed the experimental results.