Drug–plasma protein interactions have a significant impact on both pharmacokinetics and pharmacodynamics (pharmacological effects). It is thus highly interesting to evaluate this binding during the drug development process. The interaction between human serum albumin (HSA) and Nano-berberin in both the presence and absence of holo-transferrin was evaluated. Nano-berberin and holo-transferrin effectively quenched the intrinsic fluorescence of HSA via static quenching in binary and ternary systems. Synchronous fluorescence spectroscopy and three-dimensional fluorescence spectra showed that the structure of the micro-environment around the flourophore experienced a blue shift and thus a decrease in polarity. The characteristics of resonance Rayleigh scattering (RRS) spectra, the effective factor and optimum conditions of interaction were studied. It was found that the enhanced intensity of RRS was proportional to the concentration of Nano-berberin and holo-transferrin. Circular dichroism (CD) measurements indicated that Nano-berberin and holo-transferrin changed the secondary structure of HSA and that the presence of holo-transferrin led to an even more significant alteration of the secondary structure of HSA. According to Förster’s theory of resonance energy transfer, the binding distance (r) between Trp 214 and Nano-berberin in the absence and presence of holo-transferrin was predicted to be 2.25 nm and 2.03 nm, respectively, so the affinity of Nano-berberin to HSA increased in presence of holo-transferrin. Red-edge excitation shift (REES) analysis was initially applied to monitor the motion around the Trp residue in protein studies. With an increasing solute concentration, the Trp residue faced more restrictions from its surroundings. The relationship between electric charges at the protein surface was investigated by zeta potential measurements. This study indicated the influence of Nano-berberin and HSA on the surface chemistry and surface electric charge properties upon protein interaction. To acquire more information about the interaction of Nano-berberin and holo-transferrin with HSA, interaction models were generated using molecular modeling. Molecular modeling confirmed that the affinity of Nano-berberin to HSA was increased in the presence of holo-transferrin.