4,5‐Diaminofluorescein (DAF‐2) and its membrane‐permeable derivate DAF‐2 diacetate are fluorescent probes that have been developed to perform real‐time biological detection of nitric oxide (NO). Their use for intracellular imaging, however, has recently been seriously questioned and data using DAF‐2 for extracellular NO detection at low levels, as for example released from endothelial cells, are rare. Here we show that a reliable detection of low levels of NO in biological systems by DAF‐2 is possible (a) by using low DAF‐2 concentrations (0.1 μM) and (b) by subtracting the DAF‐2 auto‐fluorescence from the measured total fluorescence. The described method allows easy real‐time detection of endothelial NO formation.