The goal of this work is to develop a delivery system for histone deacetylase inhibitor (HDACi) romidepsin (ROM) using Poly(D, L-lactide-co-glycolide) as a carrier and evaluate its anti-leukemic effects. Romidepsin-loaded nanoparticles (ROM NPs) required for this purpose were fabricated using a single emulsion-solvent evaporation technique. Their physical characteristics and in vitro drug release profiles were studied, alongside biocompatibility and hemocompatibility assessments. Cell viability assays and Annexin V/Propidium Iodide (PI) staining were conducted to evaluate the anti-leukemic and apoptosis induction efficiency of ROM NPs in vitro. ROM NPs displayed a spherical shape with an average hydrodynamic size of about 149.7 ± 8.4 nm, a PDI of 0.11 ± 0.03, and a zeta potential of -25.27 ± 2.12 mV. The nanoparticles demonstrated a high encapsulation efficiency of ROM (∼93 %) and these nanoparticles effectively entered acute leukemia cells, including U937 and Jurkat. ROM NPs also exhibited a prolonged biphasic release pattern, specifically, the initial burst release phase occurred within the first 24 h, followed by a slower, sustained release. Additionally, they showed no hematological or biological toxicity, indicating their potential use for the delivery of anti-cancer drugs through the circulatory system. In tests on acute leukemia cell lines, ROM NPs showed significantly stronger anti-leukemic effects and induced apoptosis to a greater extent compared to free ROM. In summary, ROM NPs represent a promising therapy option for leukemia according to their enhanced anti-leukemic effects. Further modification of this strategy could be performed to enable target specificity, hence minimizing damage to normal cells.