Introduction: Luteolin (LUT) is natural flavonoid with multiple therapeutic potentials and is explored for transdermal delivery using a nanocarrier system. LUT loaded cationic nanoemulsions (CNE1–CNE9) using bergamot oil (BO) were developed, optimized, and characterized in terms of in vitro and ex vivo parameters for improved permeation. Materials and methods: The solubility study of LUT was carried out in selected excipients, namely BO, cremophor EL (CEL as surfactant), labrasol (LAB), and oleylamine (OA as cationic charge inducer). Formulations were characterized with globular size, polydispersity index (PDI), zeta potential, pH, and thermodynamic stability studies. The optimized formulation (CNE4) was selected for comparative investigations (% transmittance as %T, morphology, chemical compatibility, drug content, in vitro % drug release, ex vivo skin permeation, and drug deposition, DD) against ANE4 (anionic nanoemulsion for comparison) and drug suspension (DS). Results: Formulations such as CNE1–CNE9 and ANE4 (except CNE6 and CNE8) were found to be stable. The optimized CNE4 based on the lowest value of globular size (112 nm), minimum PDI (0.15), and optimum zeta potential (+26 mV) was selected for comparative assessment against ANE4 and DS. The %T values of CNE1–CNE9 were found to be ˃95% and CEL content slightly improved the %T value. The spherical CNE4 was compatible with excipients and showed % total drug content in the range of 97.9–99.7%. In vitro drug release values from CNE4 and ANE4 were significantly higher than DS. Moreover, permeation flux (138.82 ± 8.4 µg/cm2·h), enhancement ratio (8.23), and DD (10.98%) were remarkably higher than DS. Thus, ex vivo parameters were relatively high as compared to DS which may be attributed to nanonization, surfactant-mediated reversible changes in skin lipid matrix, and electrostatic interaction of nanoglobules with the cellular surface. Conclusion: Transdermal delivery of LUT can be a suitable alternative to oral drug delivery for augmented skin permeation and drug deposition.