Objective To obtain the near-infrared fluorescence image in vivo and find the relationship between the detecting depth and fluorescence probe concentration. Methods Signals of fluorescence probe in various depths of vivo and tissue phantom with cooled CCD camera were acquired. Results The fluorescence intensity informations with different fluorescence probe concentrations and depths in vivo and liquid phantom were obtained. Conclusion Relationship between fluorescence intensity,location and depth of detecting probe in vivo is found. The linear relation of fluorescence probe concentration and detecting intensity is simulated,which will be used as a reference for the experiment system.