Hypoxia in malignant tumors can have an inhibitory effect on photodynamic therapy, chemotherapy, radiotherapy, immunotherapy, and chemodynamic therapy. In previous studies, nanoparticle platforms combined with drugs or direct oxygen-carrying to increase oxygen content are usually used. Here, we designed to improve tumor hypoxia by injecting human-derived hemoglobin-based oxygen carriers (h-HBOC) synthesized from expired human blood or human placental blood, and real-time quantitative oxygen concentration monitoring was achieved with the help of photoacoustic mesofunctional imaging, and the oxygen concentration was calculated using a multispectral unmixing method. Firstly, we combined photoacoustic mesoscopy to monitor the blood oxygen concentration of the same batch of mice injected with different concentrations of h-HBOC, and verified the positive correlation between the h-HBOC concentration and the blood oxygen level without affecting the hemoglobin of the animals themselves. The relative optimal dose of h-HBOC for targeting 4T1 tumor line mice was determined to be 600 mg/kg, with the sixth hour after h-HBOC injection being the relative optimal moment of treatment. A method for determining the optimal moment of tumor microenvironment by real-time monitoring of blood oxygenation in malignant tumors has been developed to determine the optimal time of treatment for therapeutic means. This therapeutic strategy can formulate differentiated and visualized therapeutic strategies for individual individuals according to different tumor lineages and bio-individuals, bridging the individual differences and obtaining the optimal therapeutic plan. Since the improvement of tumor microenvironment by h-HBOC is sustained in the long term and can be used to improve photodynamic therapy, and chemotherapy and radiotherapy have positive effects, this strategy has great potential and application for improving multimodal treatment of malignant tumors in the future.