In hyperthermia, the accurate measurement of blood flow is regarded as a key footstep to describe physiological status of subjects since blood flow serves both to deliver oxygen and the metabolic substrates, and to carry away heat and the waste products of metabolism. To facilitate these issues we put forward a simple optical method to measure dynamical change of blood volume combining laser speckle measurement and CCD microscopic imaging technique. In this study, at first we selected six different temperatures (31°C, 33°C, 35°C, 37°C, 38°C, 39°C), followed variations of blood velocity and diameter in microvessels (15~50μm) on rat’s mesentery after 30 minutes at each temperature. Furthermore, during heated at seven higher different temperatures (41°C, 43°C, 45°C, 46°C, 49°C, 51°C, 54°C) for 30 minutes respectively, diameter and velocity were measured in vivo and real time. The results showed that changes of blood volume ranged from 68~110% when temperature altered, and the relationship between the constant value and the temperature approximated linear (k=0.05±0.005). While heated at 41°C~46°C for 30 min, velocity increased slightly but diameter and blood volume increased markedly and finally got to constants. The value of velocity increased to maximum when microvessels were heated with 49°C for 12 min. At this temperature, diameter and blood volume increased during the first 14 min, but began to decrease when heated longer. When temperature is higher than 49°C, 51°C, 54°C, velocity ,diameter and blood volume all increased at first and then decreased during 30 min and at the end of heating they were all far lower than control value. We draw our conclusion that the critical temperature is 49°C for intestine microcirculation with heating for 30 min. And the rates of thermal damage are proportion to temperature for the same heating time.