Methylphosphorylated mono-, di- and trimannosides structurally related to the lipopolysaccharide (LPS) O-antigens of Klebsiella pneumoniae of serotype O3 were synthesized and conjugated with a biotin tag. The stereo- and regioselective assembly of target carbohydrate chains was conducted using uniform monosaccharide synthetic blocks. After that, a methylphosphate group was introduced by coupling with a methyl-H-phosphonate reagent followed by oxidation and deprotection to give the target oligosaccharides. The 1H and 13C NMR spectra of the obtained compounds showed a good fit with the spectrum of the corresponding natural polysaccharide. The newly prepared biotinylated oligosaccharides along with the previously reported biotinylated glycoconjugates related to galactan I and galactan II of K. pneumoniae LPS were used for the ELISA detection of antibodies in anti-K. pneumoniae rabbit sera. Anti-O3 serum antibodies specifically recognized the synthesized oligosaccharide ligands with terminal methylphosphomannosyl residues, whereas anti-O1 serum antibodies recognized the oligosaccharide related to K. pneumoniae galactan II. The analysis of human sera from patients with confirmed Klebsiella infection also revealed the presence of antibodies against the synthesized oligosaccharides in clinical cases. Thus, the described compounds together with other Klebsiella related antigenic oligosaccharides could be potentially used as molecular probes for K. pneumoniae serological diagnostics development and strain serotyping.