In the present study, the modification of branched polyethyleneimine (b-PEI) was carried out using mesquite gum (MG) to improve its hemocompatibility to be used in biomedical applications. In the copolymer synthesis process (carboxymethylated mesquite gum grafted polyethyleneimine copolymer (CBX-MG-PEI), an MG carboxymethylation reaction was initially carried out (carboxymethylated mesquite gum (CBX-MG). Subsequently, the functionalization between CBX-MG and b-PEI was carried out using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as crosslinking agents. The synthesis products were characterized using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and thermogravimetric analysis (TGA). Thermogravimetric analysis showed that CBX-MG and CBX-MG-PEI presented a lower decomposition temperature than MG. The CBX-MG-PEI has a high buffer capacity in the pH range of 4 to 7, similar to the b-PEI. In addition, the CBX-MG-PEI showed an improvement in hemocompatibility in comparison with the b-PEI. The results showed a non-hemolytic property at doses lower than 0.1 µg/mL (CBX-MG-PEI). These results allow us to propose that this copolymer be used in transfection, polymeric nanoparticles, and biomaterials due to its physicochemical and hemocompatibility properties.