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Newport Green is a zinc-specific fluorescent dye developed to monitor cellular zinc transport. In pancreatic islets with zinc-rich β-cells, Newport Green is expected to be useful as an islet-specific indicator for live imaging. However, the low penetration of Newport Green into islets hinders clear detection. The aim of this study was to develop a practical method of live islet imaging by using surfactants to enhance the penetration efficiency.Surfactants (F127, Tween 20, and Triton X-100) were co-incubated with Newport Green for fluorescent imaging of live isolated human islet and nonislet tissues. Toxicity, enhancement of Newport Green fluorescence, and effects on specificity to islets were examined.Newport Green fluorescent intensity was increased after co-incubation with all surfactants tested (0.2–3.2 mM); however, surfactants were toxic to islets at high concentrations. Within the nontoxic range, high specificity to islets was observed when co-incubated with Tween 20 at 0.2–0.4 mM, compared with F127 and Triton X-100. This optimized range successfully distinguished islets from nonislet tissues using statistically calculated cutoff value of Newport Green fluorescent intensity.Surfactants, particularly Tween 20 in the optimized range, effectively and selectively enhanced Newport Green fluorescence in live islets without increasing islet toxicity.