The singlet oxygen quantum yield (ΦΔ) was monitored in real time through time resolved spectroscopy measurement, using gadolinium labeled hematoporphyrin monomethyl ether (Gd-HMME) as photosensitizer. According to the kinetics equations of singlet oxygen generation and reaction, ΦΔ was related to phosphorescence lifetime (τp). Through measuring τp of Gd-HMME in different oxygen conditions, the radiation transition property of first exited triplet state (T1) was monitored; combined with the triplet state quantum yield (ΦT) determined by linear fitting the ΦΔ, which was measured in different oxygen content using a relative measurement, ΦΔ can be determined in real time. The identification of anoxia during the treatment of photodynamic therapy (PDT) by this method is also presented.