Summary Background  Regional lymph node involvement is the most important prognostic factor in cutaneous melanoma. As only 20% of patients with melanoma have occult nodal disease and would benefit from a regional lymphadenectomy, the sentinel lymph node (SLN) biopsy was introduced. Near‐infrared (NIR) fluorescence has been hypothesized to improve SLN mapping. Objectives  To assess the potential of intraoperative NIR fluorescence imaging to improve SLN mapping in patients with melanoma and to examine the optimal dose of indocyanine green adsorbed to human serum albumin (ICG:HSA). Methods  Fifteen consecutive patients with cutaneous melanoma underwent the standard SLN procedure using 99mtechnetium‐nancolloid and patent blue. In addition, intraoperative NIR fluorescence imaging was performed after injection of 1·6 mL of 600, 800, 1000 or 1200 μmol L−1 of ICG:HSA in four quadrants around the primary excision scar. Results  NIR fluorescence SLN mapping was successful in 93% of patients. In one patient, no SLN could be identified using either conventional methods or NIR fluorescence. A total of 30 SLNs (average 2·0, range 1–7) were detected, 30 radioactive (100%), 27 blue (73%) and 30 NIR fluorescent (100%). With regard to the effect of concentration on signal‐to‐background ratios a trend (P = 0·066) was found favouring the 600, 800 and 1000 μmol L−1 groups over the 1200 μmol L−1 group. Conclusion  This study demonstrates feasibility and accuracy of SLN mapping using ICG:HSA. Considering safety, cost and pharmacological characteristics, an ICG:HSA concentration of 600 μmol L−1 appears optimal for SLN mapping in cutaneous melanoma, although lower doses need to be assessed.