In the tissue composition measurements in vivo based on diffuse spectroscopy, the tissue differences or variations could induce a big uncertainty. Monitoring the tissues’ optical characteristics is necessary to improve the measurement accuracy. The measurement with single light source detector separation (SDS) is not suitable to be applied widely because the absorbance of diffuse light dose not vary with SDSs linearly then. The results with one SDS hardly are transferred to that with other SDSs. In this paper, we use two SDSs to perform a differential measurement and acquire the effective attenuation coefficient (EAC) for in vivo tissues, independent of the used SDSs as a comprehensive optical property related to tissue absorption and scattering. An optical fiber leads incident light, and two detectors are designed for the two SDSs, arranged circularly with multiple optical fibers. The SDSs applied are 0.6 and 2.0 mm, respectively. The acquired EAC spectra can monitor the tissue composition variations or tissue condition fluctuations. In tralipid solutions were used to mimic tissues, and the EAC spectra of glucose concentration, hemoglobin concentration, particle density and temperature in the waveband of 1 000~1 300 nm were investigated and compared. The results show that the EAC spectra mainly demonstrate the tissue absorption caused by the variables. The EAC spectra of hemoglobin show a similar characteristic with hemoglobin’s absorption as there is a more considerable absorption at 1 000~1 200 nm and a lower at 1 200~1 300 nm. The EAC spectra caused by temperature changes appear similar to the water’s absorption change under varying temperatures. The EAC spectra caused by scattering change show less specificity, and the spectra of glucose and particle density seem similar in that they only affect scattering as glucose has less absorption at this waveband. Furthermore, the EAC spectra of human bodies are investigated and their differences between individuals or test positions, including fingers, palms, backs of hands, and lateral arms. These EAC spectra of human bodies can demonstrate the tissue differences caused by hemoglobin, water and temperature etc. In summary, this EAC spectrum measurement approach is especially suitable for in vivo tissues and provides a new way for monitoring in vivo tissues in real-time as a good indicator of absorption or scattering of the tissues.