Introduction: Growing evidence in the literature supports the accuracy of sentinel lymph node (SLN) biopsy in early-stage cervical cancer. One-step nucleic acid amplification (OSNA) is a rapid assay able to detect cytokeratin 19-mRNA in SLNs, and it can be used for intra-operative detection of low-volume metastases. The aim of this study was to evaluate the rate of low-volume metastasis in SLNs detected by OSNA in patients with early-stage cervical cancer. Secondary aims were to define the sensitivity and the negative predictive value of SLN biopsy assessed with OSNA. Methods: After IRB approval, consecutive patients who underwent surgery for International Federation of Gynecology and Obstetrics stage IA1 with lymph-vascular space involvement to IB1 between November 2017 and July 2019 and had SLN biopsy and pelvic lymphadenectomy were included. SLNs were detected with indocyanine-green cervical injection and sent intra-operatively for OSNA. Results: Eighteen patients underwent SLN assessment with OSNA and systematic pelvic lymphadenectomy in the study period. Four (22.2%) patients had unilateral and 14 (77.8%) had bilateral mapping. OSNA detected micro-metastasis in 6/18 (33.3%) patients. All micro-metastases were detected in patients with bilateral SLN mapping. The sensitivity and negative predictive value of SLN in detecting lymph node metastasis with OSNA calculated per pelvic sidewall were 85.7% and 96.1%, respectively. The false negative rate in mapped sidewalls was 14.3%. Discussion: This is the first series entirely processing SLNs for OSNA in early-stage cervical cancer. OSNA is able to intra-operatively detect low-volume metastases in SLNs. Further studies are necessary to confirm the accuracy of this technique and to assess survival implications of low-volume metastases detected by OSNA.