In this research, the preparation of biodegradable particles of keratin containing anticancer drug Doxorubicin and the evaluation of drug release were studied. For this purpose, solutions containing 16, 20 and 34% Doxorubicin in Formic acid solvent were prepared. The nanoparticles were generated by using electrospray method. Selected range for distance of the needle tip to collector was 25cm, voltage is 20 kV and feed rate was 0.1 ml/h. Diameter of the nanoparticles containing 20% anticancer drug was 50 nm. By using Fourier transform infrared spectroscopy (FT-IR) Formation of possible reaction between keratin and drug also remaining solvent in the nanoparticles was evaluated. FT-IR results showed that between Doxorubicin drug and keratin no reaction was occurred during electrospray and there was no Formic acid remained in the nanoparticle. In order to investigate the effects of adding Doxorubicin on the microstructure of keratin nanoparticles used X-ray diffraction analysis. XRD results showed that Doxorubicin as a crystal placed in the context of crystalline keratin. Drug release from nanoparticle containing Doxorubicin was evaluated in phosphate buffer with pH = 7.4. UV-Vis spectroscopy method was used to determine the amount of Doxorubicin in phosphate buffer. Results shows that drug concentration did not influence on drug release mechanism of the nanoparticles. Doxorubicin was release from nanoparticle with fick mechanism and with increasing concentrations of the drug release rate was increased. According to the results we can say keratin nanoparticles containing Doxorubicin have the ability to control the drug release and can be used as a delivery system for controlled Doxorubicin chemotherapy. Also Doxorubicin interactions with human albumin serum and keratin have been studied theoretically by using molecular docking and Ligplot and results showed two types of hydrogen and hydrophobic interactions for both. Also showed Doxorubicin drug interactions with HSA stronger than keratin that has provided favorable conditions to separate the drug from the keratin in the blood and binds to HSA. Doxorubicin interactions with FS-DNA have been studied theoretically by using molecular docking and Ligplot and Practically was investigated by using UV-Vis spectroscopy and the binding constant obtained for doxorubicin and FS-DNA from theoretical and practical was consistented.