A general, convenient, and efficient enzymatic approach for producing fluorinated mechanistic probes for sialidases and sialyltransferases is described. Other than the reported sialyltransferase inhibitor CMP-3F(axial)Neu5Ac, CIVIP-3F (equatorial)Sia derivatives (in which Sia is Neu5Ac or Neu5Gc) have been synthesized along with CMP-3F (axial)Neu5Gc and CMP-3F(axia/)KDN as novel inhibitors and important mechanistic probes for sialyltransferases. 3-Fluoro-sialosides have been enzymatically synthesized and could be used as potential inhibitors and mechanistic probes for sialidases and as probes for sialic acid-binding proteins.