Multiparametric magnetic resonance imaging (mpMRI) can guide the surgical plan during robot-assisted radical prostatectomy (RARP), and intraoperative frozen section (IFS) can facilitate real-time surgical margin assessment. To assess a novel technique of IFS targeted to the index lesion by using augmented reality three-dimensional (AR-3D) models in patients scheduled for nerve-sparing RARP (NS-RARP). Between March 2019 and July 2019, 20 consecutive prostate cancer patients underwent NS-RARP with IFS directed to the index lesion with the help of AR-3D models (study group). Control group consists of 20 patients matched with 1:1 propensity score for age, clinical stage, Prostate Imaging Reporting and Data System score v2, International Society of Urological Pathology grade, prostate volume, NS approach, and prostate-specific antigen in which RARP was performed by cognitive assessment of mpMRI. In the study group, an AR-3D model was superimposed to the surgical field to guide the surgical dissection. Tissue sampling for IFS was taken in the area in which the index lesion was projected by AR-3D guidance. Chi-square test, Student t test, and Mann-Whitney U test were used to compare, respectively, proportions, means, and medians between the two groups. Patients in the AR-3D group had comparable preoperative characteristics and those undergoing the NS approach were referred to as the control group (all p ≥ 0.06). Overall, positive surgical margin (PSM) rates were comparable between the two groups; PSMs at the level of the index lesion were significantly lower in patients referred to AR-3D guided IFS to the index lesion (5%) than those in the control group (20%; p = 0.01). The novel technique of AR-3D guidance for IFS analysis may allow for reducing PSMs at the level of the index lesion. Augmented reality three-dimensional guidance for intraoperative frozen section analysis during robot-assisted radical prostatectomy facilitates the real-time assessment of surgical margins and may reduce positive surgical margins at the index lesion. We proposed a novel technique of augmented reality three-dimensional (AR-3D) guided intraoperative frozen section (IFS) for real-time assessment of surgical margins during robot-assisted radical prostatectomy. The AR-3D guidance for IFS allows combination of the reduction of positive surgical margins at the level of the index lesion and preservation of neurovascular bundles.