This paper is the study of the diffusion constant in order to calculate the percent oxygenation and percent blood volume using reflectance light within biological tissue. The diffusion constant play major role in percent oxygenation and percent blood volume and varies with the biological material such as hemolyzed blood, whole blood, dermis and epidermis in vivo tissue. The diffusion constant can be modeled to consist of a contribution from bloodless tissue and blood present in tissue. The reflectance light for experimental are red light of 660nm, infrared light of 880nm, green light of 569nm. The correlation between the diffusion constant and biological tissue was analyzed by the intensity of reflectance light at different depth within human limb. The reflectance light was changed in response to physiological changes within biological tissue. The data for diffusion constant were obtained at different depth beneath the surface of the skin and will be utilized to amen the percent oxygenation and percent blood volume.