Albumin nanoparticles are considerable in nanotechnology due to their high solubility in aqueous media, their ability to interact with various compounds, and their ease of nanoparticle tailoring from them. Due to the undesirable effects of glutaraldehyde (GT) as a cross-linker, the aim of this study was to investigate different cross-linking agents such as tannic acid, ascorbic acid, citric acid, sorbitol, and glucose (GLU) and to optimize particle production. For this purpose, final particle size, polydispersity index, and zeta potential were measured. Nanoparticles of different cross-linkers were compared to those prepared with GT using UV–Vis and FTIR spectra. Optimal conditions were defined as 20 mg/mL bovine serum albumin (BSA) solution (2 mL), pH 9, temperature of 4 °C, ethanol as desolvation agent, and GLU as cross-linker. BSA nanoparticles were obtained 125.1 nm in size, with a polydispersity index of 0.132 at the optimum point of the desolvation process. Studying FTIR and UV–Vis results showed that cross-linking occurred in the presence of all cross-linkers. [ABSTRACT FROM AUTHOR]