Nanoparticles are used increasingly for the treatment of different disorders, including burn wounds of the skin, due to their important role in wound healing. In this study, acriflavine-loaded poly (ε-caprolactone) nanoparticles (ACR-PCL-NPs) were prepared using a double-emulsion solvent evaporation method. All the formulations were prepared and optimized by using a Box–Behnken design. Formulations were evaluated for the effect of independent variables, i.e., poly (ε-caprolactone) (PCL) amount (X1), stirring speed of external phase (X2), and polyvinyl alcohol (PVA) concentration (X3), on the formulation-dependent variables (particle size, polydispersity index (PDI), and encapsulation efficiency) of ACR-PCL-NPs. The zeta potential, PDI, particle size, and encapsulation efficiency of optimized ACR-PCL-NPs were found to be −3.98 ± 1.58 mV, 0.270 ± 0.19, 469.2 ± 5.6 nm, and 71.9 ± 5.32%, respectively. The independent variables were found to be in excellent correlation with the dependent variables. The release of acriflavine from optimized ACR-PCL-NPs was in biphasic style with the initial burst release, followed by a slow release for up to 24 h of the in vitro study. Morphological studies of optimized ACR-PCL-NPs revealed the smooth surfaces and spherical shapes of the particles. Thermal and FTIR analyses revealed the drug–polymer compatibility of ACR-PCL-NPs. The drug-treated group showed significant re-epithelialization, as compared to the controlled group.