Positron annihilation lifetime spectroscopy (PALS) is a powerful tool for the investigation of microstructure. Three main classes of materials, metals, semiconductors and polymers, have been studied by using this technique. But, relatively few investigations have been performed in the biological sciences. PALS provides important information on pore properties and free volume at the molecular level. Our PALS study showed that Yucatan miniature pig stratum corneum separated with heat and trypsin digestion had a longer positron annihilation lifetime than cyclodextrins. This indicates that the stratum corneum has larger pores and/or free volume than cyclodextrins, whose pores have a diameter of 0.5–0.8 nm and a torus height of 0.79 nm. Positron annihilation spectroscopy may be developed as a new technique for the detection of nano-pore properties and free volume in the biological sciences.