The aim of this study was to assess the superiority of ICG-99mTc-nanocolloid for the intraoperative visual detection of sentinel lymph nodes (SLNs) in vulvar squamous cell carcinoma (VSCC) patients compared to standard SLN detection using 99mTc-nanocolloid with blue dye. In this multicenter, randomized controlled trial, VSCC patients underwent either the standard SLN procedure or with the hybrid tracer ICG-99mTc-nanocolloid. The primary endpoint was the percentage of fluorescent SLNs compared to blue SLNs. Secondary endpoints were successful SLN procedures, surgical outcomes and postoperative complications. Forty-eight patients were randomized to the standard (n = 24) or fluorescence imaging group (n = 24) using ICG-99mTc-nanocolloid. The percentage of blue SLNs was 65.3% compared to 92.5% fluorescent SLNs (p < 0.001). A successful SLN procedure was obtained in 92.1% of the groins in the standard group and 97.2% of the groins in the fluorescence imaging group (p = 0.33). Groups did not differ in surgical outcome, although more short-term postoperative complications were documented in the standard group (p = 0.041). Intraoperative visual detection of SLNs in patients with VSCC using ICG-99mTc-nanocolloid was superior compared to 99mTc-nanocolloid and blue dye. The rate of successful SLN procedures between both groups was not significantly different. Fluorescence imaging has potential to be used routinely in the SLN procedure in VSCC patients to facilitate the search by direct visualization. Clinical Trial Registration: Netherlands Trial Register (Trial ID NL7443).