The interaction of methotrexate (MTX) and bovine serum albumin (BSA) was studied by fluorescence and absorption spectra. The results show that methotrexate strongly quenched the fluorescence of bovine serum albumin, and the quenching mechanism is a static quenching procedure. The binding constants and the number of binding sites between MTX and BSA at different temperatures were calculated. Furthermore, the enthalpy and entropy changes in the interaction were also obtained, and the primary binding pattern between MTX and BSA was interpreted as hydrophobic interaction.