The structural evolution and nanoscale properties of PLLA nanofiber during the early quenching period in a thermally induced phase separation process have been investigated. The PLLA/THF solution was prepared at 60°C and quenched in a −24°C refrigerator for different times to become a gel. The morphology, phase transition, crystallization behavior, hydrophobicity, variation of chain conformation and adhesion force were studied by using SEM, WAXD, ATR-FTIR, DSC, contact angle measurement, AFM and force spectroscopy measurements. The initial phase separation resulted in an amorphous gel with the condensation of nanoparticles, followed by the nucleation of PLLA crystals. The α-form crystal appeared after quenching for 1min, corresponding to a gel temperature around 15°C. After quenching for sufficient time (longer than 3min) and the gel temperature decreasing to below 0°C, a nanofibrous architecture was formed with a limited disorder α′-form crystal. With the extending of the gelation time, the degree of crystallinity of the matrix increased and conformational transformation of the polymer chains proceeded with more closely packed polymer segments that restricted the backbone COC vibration and strengthened interchain interaction of the CO bonds. The surface morphology and structural evolution led to the increase in the hydrophobicity as well as nanoscale mechanical property of the nanofibers. AFM and force spectroscopy measurements of 2-D samples on the glass slide showed that the nanofiber formation seemed to initiate from a central nuclei and grew radially outward and larger fibers were assembled laterally with a bunch of thinner nanofibers. The adhesion force increased with the gelation time which suggested that the structural evolution of PLLA chains conformation and the chain packing has a direct functional consequence in the nanoscale mechanical property.