Development of biosimilars is costly, where glycan analysis is a significant constraint on time and money. This paper provides an in-depth characterisation of several novel recombinant prokaryotic lectins (RPLs), developed through directed evolution, displaying specific binding activities to α-mannose, β-galactose, fucose and sialic acid residues, tested against major biosimilar targets. The binding characterisation of all lectins was performed employing the principles of bio-layer interferometry (BLI), with help of the streptavidin-coated sensor with the biotinylated lectins. The binding activity of the RPLs and the specificity to a broad range of glycoproteins and glycoconjugates were evaluated and compared to those of equivalent plant-derived lectins. While exhibiting better or similar specificity, RPLs displayed significantly better binding in all cases. The binding mechanisms are explained with particular focus on the role hydrogen bonding plays in the change of specificity for a galactose specific lectin. Furthermore, different sets of RPLs and their plant equivalents were assayed against the different glycoprotein targets to evaluate the analytical parameters of the lectin-glycoprotein interaction. The obtained LoDs reached by the RPLs were lower than those of their plant counterparts apart from one, exhibiting RPL:PL LoD ratios of 0.8, 2.5, 14.2 and 380 for the sets of lectins specific to fucose, α-mannose, β-galactose and sialic acid, respectively. Such enhancement in analytical parameters of RPLs shows their applicability in protein purification and as bioanalytical tools for glycan analysis and biosensor development.