In this contribution, we study the effect of confinement on the ultrafast electron transfer (ET) dynamics of riboflavin binding protein (RBP) to the bound cofactor riboflavin (Rf, vitamin B2), an important metabolic process, in anionic sodium bis(2‐ethylhexyl) sulfosuccinate reverse micelles (AOT‐RMs) of various hydration levels. Notably, in addition to excluded volume effect, various nonspecific interactions like ionic charge of the confining surface can influence the biochemical reactions in the confined environment of the cell. To this end, we have also studied the ET dynamics of RBP–Rf complex under the confinement of a cationic hexadecyltrimethylammonium bromide (CTAB) RMs with similar water pool size to the anionic AOT‐RMs towards simulating equal restricted volume effect. It has been found that the spatial confinement of RBP in the AOT‐RM of w0 = 10 leads to the loss of its tertiary structure and hence …