A new method of pain measurement using scattering spectrum with a small source-detector separation is proposed. First, the relationship between the change of oxy-hemoglobin and deoxy-hemoglobin concentrations and the change of scattering spectrum intensity sampled through an optical probe with a small source-detector separation is established based on the modified Beer-Lambert’s law. The formula calculating results are compared with those measured by tissue oximeter in experiments using Intralipid mixed with blood. Secondly, the formalin solution was injected to seven rats’ hind paws to create pain stimulation. The spectrum system with a small source-detector separation was used to measure the scattering spectrum of the anterior cingulate cortex (ACC) of rats before, and after injection, meanwhile, the local field potential of ACC before and after injection were acquired by the bioelectrical signal acquisition system. Finally, the change of oxy-hemoglobin, deoxy-hemoglobin and total hemoglobin concentration were calculated. The power spectrum intensity of β wave (13~30 Hz) was obtained every 10 seconds, respectively. The changes in these parameters before and after injection were analyzed. The results showed that the values of the oxy-hemoglobin and deoxy-hemoglobin concentration change calculated by the formula are in good agreement with those measured by the Oximeter in the liquid model experiment, in which the average relative error with the standard deviation is 14%±5%. Therefore, the analysis of the change of oxy-hemoglobin and deoxy-hemoglobin concentration by the spectrum system with a small source-detector separation could have high accuracy. In the pain stimulation experiments of rats, the oxy-hemoglobin concentration of ACC increased rapidly for a short time within 5 minutes after the injection of formalin and then decreased monotonously. Meanwhile, the deoxy-hemoglobin concentration decreased in a short time within 5 minutes after injection and then increased monotonously. There was no significant change in total hemoglobin concentration after injection. The local field potential power of ACC gradually increased after the injection and reached the peak value about 20 minutes after injection, and then remained near the peak value, which was significantly different from that before injection. It can be confirmed that the oxy-hemoglobin and deoxy-hemoglobin concentration changes were correlated highly with ACC’s functional activation. It can be seen that the local scattering spectrum with a small source-detector separation can be effectively used to mark the functional activation and analyze the neural-vascular coupling mechanism of the nuclei related to pain, which provides an effective new way to improve the understanding of cerebrovascular function related to pain and pain measurement in small animal models.