The authors investigated two-photon nanolithography (TPNL) of positive AZ 5214E photoresist thin film with a spatial resolution at nanoscale. A continuous trench with average width of 101 nm was obtained, and a trench with several break points but feature width of 19 nm was fabricated by further increasing the scanning speed. Hole arrays composed of holes with diameters of about 451 nm and period of 800 nm were also fabricated with this photoresist. These results not only reveal that the TPNL of positive photoresist can obtain similar spatial resolution to that of negative photoresists, but also have extensive application prospection in fabrications of integrated circuits, microfluidic devices, surface-enhanced Raman scattering (SERS) substrates, catalyst, and biosensor substrates.