The study of interaction between spirooxindole-annulated thiopyran derivatives (STP) and bovine serum albumin (BSA) was investigated using multi-spectroscopic and docking method. The intrinsic fluorescence of BSA could effectively quenched by STP through dynamic quenching. The thermodynamic parameters suggested that hydrogen bonds and van der Waals forces played a key role in stabilizing the BSA–STP complexes. According to Forster non radiation energy transfer theory (FRET) the average binding distance (r) between STP and BSA were found to be < 7 nm. Furthermore, UV-visible and circular dichroism results indicated that in presence of STP secondary structure of BSA changed. Theoretical docking study of the interaction of BSA and STP also supported the experimental results.