The effect of reverse micelle environment on the binding mechanism of 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) with Bovine Serum Albumin (BSA) compared with that in buffer solution has been investigated in this paper with the help of steady state and time-resolved emission spectroscopy along with molecular docking to have a correct picture about binding. The binding of DASPMI with attachment efficiency of 30% and 70% at site 1 (subdomain HA) and site 11 (subdomain MA) of BSA, respectively, in buffer solution gets reversed inside a reverse micelle. The bigger cavity size of site IT in buffer solution ushers the dye with increased attachment efficiency and in reverse micelle change in pi-stacking and hydrophobic interaction control the attachment efficiency. The calculated Forster distance gets curtailed as the environment changes from buffer to reverse micelle. The binding becomes stronger with a smaller gap between the probe and Trp-214 inside the reverse micelle than that in buffer solution.