The hemoglobin was extracted from the blood which was provided by the healthy volunteers and the impact of the pH on hemoglobin oxygen binding capacity was studied with microscopic Raman spectroscopy. The results indicated that: under the excitation light of 514.5 nm, with the reducing of the oxygen partial pressure (PO2), the Raman peak intensity at 1 375, 1 562, 1 585 and 1 638 cm-1 of the control hemoglobin (pH 7.4) reduced gradually, among which, the change of the 1 375 and 1 638 cm-1 were the most significant and had a good relevance with the PO2. The curves were plotted by regarding the PO2 as the x-axis and the Raman absolute intensity as the y-axis, and the relationship between hemoglobin Raman absolute intensity of the 1 375 and 1 638 cm-1and their related PO2 levels when the pH was 5.7, 7.4 and 8.0 respectively were analyzed. The data was well linear fitted and the fitting equation was obtained. The relationship of the slope (Raman intensity/PO2 level) among them were K8.0＞K7.4＞K5.7, indicating that the lower the pH, the easier the release of the oxygen molecules. It was showed that the Raman spectroscopy technique could be used to detect the oxygen binding rate of hemoglobin quantitatively, and the effect of the PH on oxygen binding state of hemoglobin could be observed, which could provide a new method and make a foundation for the monitoring of the PO2 levels in the blood, as well as the research on the regulatory factors of the blood oxygen affinity, such as H+ and CO2.