Studies on interactions between drugs and carrier proteins in blood is important for elucidating the transport, distribution, metabolism and efficacy of drugs in vivo. In this report, the fluorescence quenching mechanism of anti-HIV drug stavudine (D4T) with human serum albumin (HSA), bovine serum albumin (BSA) and hemoglobin (Hb) were determined with investigatingsteady state fluorescence, UV-Vis absorption spectra , kinetic transient emission spectra and cyclic voltammetry. The binding constant Ka (the size of the order of Ka is Hb＞HSA＞BSA) and binding sites n (n=1) of D4T binding with carriers were obtained in different temperatures (300, 310 and 320 K). Analysis of thermodynamic parameters ΔH, ΔS and ΔG of binding process, we can know that these three types of blood proteins are the ΔG＜0 and ΔH＜0, illustrating that combination of d4T and carriersare a spontaneous exothermic process, also by the ΔH＜0 and ΔS＜0, we can deduce that the binding force between D4T and HSA, BSA and Hb are hydrogen bonds and van der Waals force. The possibility of energy transfer and binding distance (R0 and r) between donor (proteins) and acceptor (D4T) were obtained according to non-radiative energy transfer theory (FRET). The r is less than 7 nm and 0.5R０＜ｒ＜1.5R０ show that from HSA, BSA and Hb to D4Tthe energy transfer occurs a great possibility. Simultaneously using synchronous fluorescence, three-dimensional fluorescence and circular dichroism spectroscopy, we can draw that the secondary structure are not affected and the changes of the tertiary conformation are not very large in the process of D4T combining with carriers (HSA, BSA and Hb). All these experimental results suggest that HSA, BSA and Hb three kinds of blood proteins can be used as good carriers in delivering D4T to the target location. These results provide an experimental basis for further study on the application of D4T in drug design and anti HIV effect.