Lactose operon contains two CRP binding sites at promoter(CRP1 site) and operator(CRP2 site) regions at lac operon. CRP protein can bind to both sites with the different binding affinity. CRP1 site, major CRP binding site, acts the transcription activation with the fully unknown mechanism by binding of CRP. In this study, the binding affinities of CRP1 site and CRP2 site were measured with the fluorescein-labeled oligomers, which contain CRP1 site and the three different spacing sequences between GTGA and TCAC at CRP2 site. Results showed that CRP:cAMP complex bound to CRP1 site 3 times more strongly than CRP2 site and the base spacing between GTGA and TCAC was not the only factor to affect the binding affinity of CRP to CRP2 site.