The aim of this study was to create nanoparticles of human serum albumin immobilized with anti-TB drugs (rifampicin, isoniazid) using the desolvation method. Central Composite Design (CCD) was applied to study the effect of albumin, urea, L-cysteine, rifampicin and isoniazid concentration on particle size, polydispersity and loading degree of the drugs. The optimized nanoparticles were spherical in shape with an average particle size of 216.7 ± 3.7 nm and polydispersity of 0.286 ± 4.9. The loading degree of rifampicin and isoniazid in the optimized nanoparticles were 44% and 27%, respectively. The obtained nanoparticles were examined by Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC); the results showed the absence of drug–polymer interactions. The drug release from the polymer matrix was studied using dialysis membranes.