Bis-PEG3-NHS Ester is widely used in bioconjugation for crosslinking biomolecules such as proteins, peptides, and antibodies. Its primary application lies in the development of antibody-drug conjugates (ADCs), where it serves as a spacer to link the antibody to a cytotoxic drug, improving solubility and reducing aggregation. The PEG3 spacer enhances hydrophilicity and provides flexibility, which can improve binding efficiency and stability of the conjugate.

It is also employed in surface modification of nanoparticles and biosensors, where controlled immobilization of biomolecules is required. The NHS ester groups react efficiently with primary amines (e.g., lysine residues) under mild conditions, enabling site-specific conjugation. Due to its homobifunctional structure, it can bridge two amine-containing molecules, making it useful in constructing protein-protein or protein-polymer complexes.

Additionally, Bis-PEG3-NHS Ester is applied in proteomics research for protein labeling and crosslinking studies, helping to elucidate protein interactions and structural dynamics. Its water-soluble nature and biocompatibility make it suitable for use in physiological environments without the need for organic co-solvents.