Direct site-specific methods of chemical protein functionalization are highly desirable. However, such methods are particularly challenging to develop due to the tremendous difficulty of chemically differentiating the same amino acid type at different protein sites. Herein, we proposed “metal binding targeting” strategy and developed Copper Assisted Sequence-specific conjugation Tag (CAST) to achieve single site-specific protein conjugations. CAST possesses superior reaction kinetics with a rate constant of 8.1 M^-1 s^-1 in aqueous buffer. Importantly, CAST conjugation can be employed as a universal method for efficient and quantitative payload attachment on different proteins. We also highlight that an antibody drug conjugate prepared using CAST is highly stable in plasma and exhibits potent efficacy in vitro and in vivo.