With the widespread application and development of photodynamic therapy (PDT) in cancer treatment, how to accurately quantify photodynamic dose for real-time adjustment and optimization of dose has become a hot topic in PDT research. This study utilized singlet oxygen probes to investigate the photophysical process of zinc phthalocyanine (ZnPc) generating singlet oxygen (1O2) under light excitation conditions. We analyzed the absorbance changes of the mixed solution of ZnPc and 1,3-diphenylisobenzofuran (DPBF) over time under varying light fluence rates. The experimental results indicated that after 90 s irradiations at light fluence rates of 40 mW/cm², 30 mW/cm², 20 mW/cm², and 10 mW/cm², the consumptions of DPBF were 77.5%, 62.7%, 50.2%, and 32.4%, respectively. The total productions of 1O2 were calculated to be 1375.8 μM, 882.5 μM, 631.7 μM, and 364.1 μM, respectively. This successfully linked the concentration changes of the singlet oxygen probe with the total production of singlet oxygen, enhancing the accuracy of photodynamic dose research.