In this study, a bundle of poly(l-lactic acid) (PLLA) hollow nanofibers was prepared through anodic aluminum oxide (AAO) membrane wetting method. The infiltrated PLLA in the nanopores of AAO membrane were isolated via etching of the membrane using phosphoric acid, preserving their original nanostructure. The outer diameter of isolated PLLA nanofibers and wall thickness were found to be 235 ± 17 nm and 38.5 ± 9 nm, respectively. The differential scanning calorimetry analysis showed that the hollow PLLA nanofibers exhibited lower melting and crystallization temperatures than the bulk PLLA sample. Furthermore, the wide-angle X-ray diffraction measurement suggested that PLLA crystallized preferentially along the 110/200 plane direction due to confinement effect of the AAO walls. Such hollow PLLA nanofibers may have potential application as a drug delivery system.