Abstract: At pH9.25 Britton-Robinson buffer solution, the interaction between Chlorophosphonazo Ⅰ（CPⅠ）and proteins has been studied by using fluorescence spectroscopy method. The emission spectrum of CPⅠ-BSA shows a bathochromic shift from 578nm to 610nm. The optimum condition of fluorimetric determination of protein was studied. The linear equation was △F=20.8C-6.8 (g/mL). The detection limit of this method was 0.031g /mL with the linear range of 0.04~30.0g /mL（r=0.9956）. The developed method was successfully applied to the determination of protein in sample. In addition, the formation constant at different temperatures and the thermodynamic functions(such as G, H and S) of CPⅠwith BSA were obtained. the mechanism of the fluorescence quenching was also explored, it is considered that the quenching is mainly due to generating quenchable complex and that interaction of CPⅠand proteins was static electricity forces.