The abnormal accumulation and deposition of islet amyloid polypeptide (IAPP) are important causes of type-2 diabetes mellitus (T2DM). The common anti-amyloid strategy employs inhibitors to prevent the formation of oligomers and the cytotoxicity caused by them, thus reducing the production of amyloid fibres. Therefore, the real characterization of the oligomers formed at the early stage of aggregation is crucial to understanding the structure of IAPP and the drug development of T2DM. For the first time, in this study, native cold-spray ionization mass spectrometry (CSI-MS) technology was employed to characterize the oligomers. It was found that CSI was more suitable for the determination of these unstable species compared to traditional ESI-MS. The ionic strengths, organic solvent and pH all had effects on the characterization of oligomers and the stability of protein conformation. The MS/MS experiments showed that odd-charge dimer ions were mainly composed of two monomer products. Moreover, a CSI-MS method for the rapid screening of IAPP–inhibitors was established and two of the most potential inhibitors (rutin and quercitrin) were screened from a series of flavonoids. Then, the structure–activity relationship and the mechanism between flavonoids and IAPP were studied. The results showed that 3-OH and sugar chains play a vital role and hydrogen bonds are the main binding force. We further confirmed that rutin and quercitrin could effectively inhibit the fibre formation of IAPP by fluorescence and TEM experiments. This study provides a new insight for analyzing the structure of IAPP and screening potential drugs for T2DM.