Various cardiotoxin (CTX) were reported having the ability to interact with heparin molecule. The heparin-derived tetrasaccharides were obtained and studied the binding properties of CTX A3 from Taiwan cobra. In this study, we have used 1D selective excitation NMR methods to identify the sequences of tetrasaccharides and binding affinities toward to CTX A3, instead of using traditional purification. 1D selective methods ascertained the presence of three major compounds that are dHex2S-GlcNS6S-IdoA2S-GlcNS6S, dHex2S-GlcNS6S-GlcA-GlcNs6S and dHex2S-GlcNS6S-IdoA2S-GlcNS. Binding affinities between these three compounds and CTX A3 were analyzied by comparing NOE building-up curve and T1 values. In addition, the proton-proton couping constant and transfer NOEs were measured for monitoring the conformational change of tetrasaccharides. The bound structure of tetrasaccharides after binding to A3 is reported after using a computer program, COmplete Relaxation and Conformational Exchange Matrix (CORCEMA).