The aim of the present study is the preparation and physicochemical characterization of chlorambucil-loaded poly(butylcyanoacrylate) nanoparticles. Chlorambucil is a lipophilic drug, which is used clinically against chronic lymphocytic leukemia, lymphomas, and other types of malignant diseases. However, the chlorambucil use is limited by its chemical instability and toxic side effects. A promising approach to circumvent these drawbacks is the entrapment of chlorambucil in a suitable nanosized carrier. Toward this goal, poly(butylcyanoacrylate) nanoparticles meet the requirements for a drug carrier system due to their biocompatibility, biodegradability, low toxicity, and ability to overcome the multidrug resistance in cancer cells. We prepared chlorambucil-loaded poly(butylcyanoacrylate) nanoparticles, which are characterized for chemical composition, particle size, drug content, and drug release. It is expected that the utilization of poly(butylcyanoacrylate) nanoparticles as a drug carrier system will pave the way toward more effective use of chlorambucil in the treatment of cancer.