The interaction between novel spiro thiazolo[3,2-a][1,3,5]triazines (NSTT) and bovine serum albumin (BSA) was investigated using fluorescence and ultraviolet spectroscopy at different temperatures (302 and 310 K) under imitated physiological conditions. The experimental results show that the fluorescence quenching mechanism between NSTT and BSA is by a static quenching mechanism. The binding constant (K (a)) and number of binding sites (n) between NSTT and BSA at different temperatures were obtained. Negative values of a dagger GA degrees, a dagger HA degrees, and a dagger SA degrees indicate that the interaction between NSTT and BSA is driven by hydrogen bonds and van der Waals forces. Using the Forster non-radiation energy transfer theory, the binding distance between BSA and NSTT was calculated. These results provide valuable information on the interaction between NSTT and BSA as well as the influence of substituent groups on the interaction.