Increasing knowledge on the biomedical applications of nanoemulsions has necessitated the fundamental understanding behind protein-nanoemulsion interaction. Spectroscopic studies on the binding strategies of biomolecules with serum albumins have been routine for many years to elucidate their binding mechanism cum conformational changes. In the present study, we have investigated the effect of neem oil nanoemulsion of different concentration towards bovine and human serum albumin. Spectroscopic methods such as fluorescence emission, synchronous and three-dimensional spectra, UV-Visible spectroscopy, FTIR and circular dichroism techniques were used. Decrease in fluorescence intensity and controversial hyper-chromicity observed in the UV-Visible spectra of albumins confirmed the ground state complex formation between neem oil nanoemulsion and protein fluorophores. Complex attained remains non-fluorescence and static. Reduced intensity of the peak 1 and peak 2 regions of three-dimensional spectra have recommended the possible effect of nanoemulsion towards the aromatic micro-environment of tryptophan and tyrosine residues. Minor shifts observed in the amide frequency of the FTIR bands at around 1600 cm- 1 corresponds to the possibilities of variation in the secondary alpha-helical structures of biomolecules on interface with nanoemulsion. Decrease in the far UV-CD absorbance cum shift in the dichoric band of molecules at 208 and 222 nm has proportionate the loss of alpha-helical structures. Prominent changes observed in the near UV-CD spectra of biomolecules have also suggested the possible alterations towards tryptophan residues of peptide backbone. © 2015 Elsevier B.V.