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 (pO 2 ) 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 pO 2 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 pO 2 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.