Unicellular diazotrophs are recognized as important contributors to the aquatic nitrogen cycle. Yet, quantifying the number of aquatic diazotrophs, especially the unicellular fraction, is highly challenging and relies mostly on microscopical and molecular approaches. In this study, a new flow cytometry-based method was developed to quantify unicellular diazotrophs by immunolabeling the nitrogenase enzyme. The quantification method was initially developed using a diazotrophic monoculture ( Vibrio natriegens ) and verified by different controls as well as various auxiliary approaches such as N 2 fixation and confocal laser scanning microscopy. It was evident that only 15–20% of the total number of V. natriegens cells synthesized the nitrogenase enzyme, even though the media was N-limited and anaerobic. This approach was further tested on field samples collected from marine and freshwater environments. Overall, the number of unicellular diazotrophs measured in various aquatic environments ranged from 1 to 6 × 10 7 cells L −1 . However, the fraction of these diazotrophs compared to total bacteria was significantly lower in the Eastern Mediterranean Sea (0.1%) than that collected along the Jordan River (4.7%). Nevertheless, N 2 fixation rates per cell were highest in the Mediterranean Sea (~ 90 attomole N cell −1 d −1 ), while lower in the lake and the river measured herein (1.4 to 0.3 attomole N cell −1 d −1 , respectively). Following the above, this direct quantification approach can provide new insights on the number and specific contribution of unicellular diazotrophs to total N 2 fixation in marine and freshwater environments.