Cysteine (Cys)-specific bioconjugation has wide-spread applications in the synthesis of protein conjugates, particularly for the functionalization of antibodies.Here, we report the discovery of trans-styryl sulfonyl fluoride (SSF) as a near-perfect Michael acceptor for Cys-specific protein bioconjugation.Compared to maleimides, which are predominantly used, SSF exhibited better chemoselectivity, self-stability, and conjugate stability while maintaining comparable reactivity.Using SSF-derived probes, proteins can be readily modified on the Cys residue(s) to install functionalities, for example, fluorescent dyes, toxins, and oligonucleotides, without influencing the activity.Further applications of SSF-derived serum-stable antibody-drug conjugates and PD-L1 nanobody-oligo conjugates demonstrate the great translational value of SSF-based bioconjugation in drug development and single-cell sequencing.