The invention relates to a novel biotin reagent, methods of preparing and labelling same and uses thereof. More specifically, the invention relates to a novel reagent of biotin, biotin chloride or chloride of 5-((3aS,4S,6aS)-2-oxo-hexahydro-1H-tieno[3,4-d]imidazole-4-yl)pentanoyl, as well as the one-step preparation and the one-step labelling of same. Both the biotin chloride and the biotinylate marker can be bound to a large number of biomolecules (proteins, enzymes, peptides, oligosaccharides or lipids), thereby enabling the use of same in studies analysing the involvement of said biomolecules in biological processes and in the preparation of diagnostic systems.