Nanoparticles (NPs) have been used in various applications such as biotechnology, nanomedicine, and drug delivery systems. Many nanoparticle drug delivery systems have been promoted for cancer treatment, and numerous materials have been investigated to use as drug delivery agents to enhance the therapeutic efficiency and safety of anticancer drugs. Albumin is a natural biopolymer and the most abundant protein in blood plasma. Due to its versatile binding capacity of widespread therapeutical drugs, albumin becomes an ideal material to obtain nanoparticles. In this study, the ionic liquid (IL) based emulsification methods were investigated. Instead of classical toxic and volatile solvents, using ILs in microemulsions, environment-friendly media were received to synthesize bovine serum albumin (BSA) NPs. In order to obtain BSA NPs, high-speed homogenizer processing was applied by following crosslinker addition. The IL microemulsions are a thermodynamically stable colloidal dispersion containing spherical droplets (W/IL or IL/W) in submicron sizes that act as nanoreactors for NP formation. Chlorambucil (CHL) was used as a model drug to investigate drug loading and releasing kinetics of BSA NPs as a drug delivery candidate. Results showed that chlorambucil loading capacities and release kinetics depended on the synthesized medium such as anion-type of ILs and surfactants. CHL loaded to the BSA NPs synthesized in hydrophilic IL BmimBF4 in relatively higher amounts and released in the same trend. In addition, the cell viability effect of CHL-loaded BSA NPs synthesized in different types of ILs were investigated. The CHL-loaded BSA NPs synthesized in BmimOTf and BmimPF6 reduced the cancer cell viability more than the used same dose of free CHL.