Proteins' C-terminal hydrazides are useful for bioconjugation and constructing proteins from multiple fragments via native chemical ligation. To improve the preparation of C-terminal hydrazides， we developed an efficient intein-based preparation method using thiols and hydrazine to accelerate formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield but also improves biocompatibility. We expanded the method's versatility by employing Pyrococcus horikoshii RadA split intein， which can accommodate a broad range of extein residues before the site of cleavage. Besides， the use of split RadA helped prevent intein proteolytic cleavage in vivo. It also offers greater control over initiation of the intein cleavage reaction. We demonstrate the preparation of C-terminal hydrazide in model proteins as well as its conjugation with an aldehyde.