A new oxime ligand named isophthalaldoxime (1) was synthesized by the addition of hydroxylamine hydrochloride to isophthalaldehyde. Then, a four nuclear palladacycle complex {Pd₄[(N,C)(NOHCHC₆H₂CHNOH)(μ-Cl)₂]₂} (2) was prepared and fully characterized using elemental analysis, FT-IR, and NMR spectroscopy. A desolvation method was employed for the synthesis of protein nanoparticles using bovine serum albumin (BSA). Then, complex (2) was loaded on the prepared nanoparticles with an optimized entrapment efficiency (EE) of 97.78% and loading capacity (LC) of 22.68%. FT-IR spectroscopy and circular dichroism (CD) were used for spectral studies. Field emission scanning electron microscopy (FESEM) and dynamic light scattering (DLS) measurement was applied for structural studies on the morphology, size, and zeta potential of BSA nanoparticles (BSA-NPs) and complex-loaded nanoparticles ((2)@BSA-NPs). The results showed the stability and acceptable size of nanoparticles prepared for the in vitro drug release experiment. Therefore, the in vitro release of (2) from BSA-NPs was studied using the dialysis bag method. In addition, the release mechanism was investigated by mathematical methods and the results showed that phase I and II of the release process followed Korsmeyer–Peppas and Higuchi models, respectively. Finally, the in vitro cytotoxicities of the synthesized nanoparticles, palladium complex, and complex-loaded nanoparticles were carried out against A549 human lung carcinoma and K562 human leukemia cell lines using MTT colorimetric assays.