The interaction between chitosan macromolecular prodrug of 5-fluorouracil (CS-5-FU) and bovine serum albumin (BSA) was investigated by fluorescence quenching technique under the simulative physiological conditions. The quenching mechanism of fluorescence of BSA by CS-5-FU was discussed. The binding constants and the number of binding sites were measured. The values of thermodynamic parameters ΔG, ΔH, and ΔS were calculated at three different temperatures. Apparent binding constant and binding sites were obtained. The results showed that the interaction between CS-5-FU and BSA agrees well with the site-binding model.