The objective of the present study was to use hollow microneedles for enhancing the transdermal delivery of Bovine Serum Albumin (MW~66,000 Da)-Fluorescein Isothiocyanate (BSA-FITC) conjugate, a hydrophilic large molecular compound. BSA-FITC was successfully loaded into the lower epidermis as well as the superficial dermis of the skin of neonatal pig by a hollow microneedle. The higher the volume of BSA-FITC solution injected, the higher the amount and the faster the BSA-FITC release rate from skin. These release profiles of BSA-FITC solution were expressed by Fick’s law of diffusion. These results suggest the utilization of hollow microneedle to enhance transdermal delivery of protein and provide useful information for designing an effective hollow microneedle system.