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Nitric oxide (NO) exhibits both pro‐ and anti‐tumor effects. Therefore, real‐time in vivo imaging and quantification of tumor NO dynamics are essential for understanding the conflicting roles of NO played in pathophysiology. The current molecular probes, however, cannot provide high‐resolution imaging in deep tissues, making them unsuitable for these purposes. Herein, we designed a photoacoustic probe with an absorption maximum beyond 1000 nm for high spatial quantitative imaging of in vivo tumor NO dynamics. The probe exhibits remarkable sensitivity, selective ratiometric response behavior, and good tumor‐targeting abilities, facilitating ratiometric imaging of tumor NO throughout tumor progression in a micron‐resolution level. Using the probe as the imaging agent, we successfully quantified NO dynamics in tumor, liver and kidney. We have pinpointed an essential concentration threshold of around 80 nmol/cm3 for NO, which plays a crucial role in the "double‐edged‐sword" function of NO in tumors. Furthermore, we revealed a reciprocal relationship between the NO concentration in tumors and that in the liver, providing initial insights into the possible NO‐mediated communication between tumor and the liver. We believe that the probe will help resolve conflicting aspects of NO biology and guide the design of imaging agents for tumor diagnosis and anti‐cancer drug screening. [ABSTRACT FROM AUTHOR]