Pump-probe-based photoacoustic tomography (PP-PAT) is an innovative and promising molecular imaging technique. In this study, we utilized PP-PAT for the first time, to the best of our knowledge, to monitor the dynamics of oxygen partial pressure (pO2) within murine tumors during photodynamic therapy (PDT) with methylene blue (MB). We developed, to our knowledge, a novel two-step fitting method to simultaneously map both the pO2 and the MB concentrations and implemented it with mexCuda to accelerate the pixel-wise-based calculation. The results demonstrated a penetration depth of up to 5 mm and revealed a significant decrease in pO2 during the PDT process, consistent with existing research findings. This study suggests that PP-PAT has the potential to become a valuable tool for intraoperative monitoring of PDT, thereby enhancing therapeutic efficacy.