Polyphenols such as epigallocatechin gallate (EGCg) may have roles in preventing some chronic diseases when they are ingested as components of plant-based foods and beverages. Human serum albumin (HSA) is a multi-domain protein that binds various ligands and aids in their transport, distribution, and metabolism in the circulatory system. In the present study, the HSA-EGCg interaction in the absence or presence of fatty acid has been investigated. Förster resonance energy transfer (FRET) was used to determine inter- and intra-domain distances in the protein with and without EGCg and palmitic acid (PA). By labeling Cys-34 with 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CPM), the distance between Trp-214 at domain IIA and CPM-Cys-34 at domain IA could be established. A small amount of PA decreased the distance, while a large amount increased the distance up to 5.4 Å. EGCg increased the inter-domain distance in HSA and HSA-PA up to 2.8 and 7.6 Å, respectively. We concluded that PA affects protein conformation more significantly compared to EGCg. Circular dichroism (CD) established that EGCg affects protein secondary structure more significantly than PA. PA had little effect on the α-helix content of HSA, while EGCg decreased the α-helix content in a dose-dependent fashion. Moreover, EGCg decreased α-helix content in HSA and HSA-PA to the same level. Dynamic light scattering (DLS) data revealed that both PA and EGCg increased HSA aggregation. EGCg increased HSA aggregation more significantly and promoted formation of aggregates that were more heterogenous. Any of these effects could impact the ability of serum albumin to transport and stabilize ligands including EGCg and other polyphenols.