A fluorescent neuraminidase (NA) assay has been developed; 20 samples in five replicates could be analyzed at the same time, allowing us to study the kinetics of the enzyme-substrate interaction. The specificities of six influenza H1N1 virus NAs for BODIPY-labeled 3´SiaLac, 3´SiaLacNAc, SiaLe(c), SiaLe(a), 6´SiaLac, and 6´SiaLacNAc were evaluated. The duck virus NA hydrolyzed 6´SiaLac and 6´SiaLacNAc 50 times more slowly than 2-3 isomers. Swine viruses digested SiaLe(a) and 2-6 sialosides 20 times more slowly than 2-3 trisaccharides. For the human viruses, the difference between 2-6 and 2-3 oligosaccharides desialylation efficiency did not exceed five times; notably, the inner core of 2-3 sialosaccharide was discriminated. The results are evidence that influenza virus NAs can distinguish substrate structure at the tri- and tetrasaccharide level.