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We proposed a two-step poling technique to fabricate nanoscale domains based on the anti-parallel polarization reversal effect in lithium niobate on insulator (LNOI). The anti-parallel polarization reversal is observed when lithium niobate thin film in LNOI is poled by applying a high voltage pulse through the conductive probe tip of atomic force microscope, which generates a donut-shaped domain structure with its domain polarization at the center being anti-parallel to the poling field. The donut-shaped domain is unstable and decays with a time scale of hours. With the two-step poling technique, the polarization of the donut-shaped domain can be reversed entirely, producing a stable dot domain with a size of tens of nanometers. Dot domains with diameter of the order of ∼30 nm were fabricated through the two-step poling technique. The results may be beneficial to domain-based applications such as ferroelectric domain memory.