Transdermal drug delivery, transporting the drug molecules through epidermis to dermis, has been extensively investigated but not studied in dynamic detail. The objective of this study is to monitor the dynamical changes of drug permeation and that of polarization of skin tissue with oleic acid treatment. We utilize two-photon fluorescence microscopy (TPM) to investigate the dynamics of transdermal drug delivery in skin excised from the abdominal region of euthenized nude mice with sulforhodamine B (SRB) modeling as a drug and Laurdan serving as a polarity indicator. The treatment of oleic acid increases the permeation rate of SRB, quickly reaching to the steady state of permeation. Increases in polarity within the skin tissue (in both intercellular and intracellular region of stratum corneum) are observed in SRB permeation enhanced by oleic acid treatment. TPM has successfully demonstrated the ability to study spatial distribution of transport dynamics in oleic acid-enhanced transdermal delivery.