This study introduces semiconducting polymer nanoparticles (SPNs) as a new class of contrast agents for photoacoustic (PA) molecular imaging in living mice. SPNs, derived from photovoltaic semiconducting polymers, exhibit strong NIR light absorption and produce higher PA signals compared to single-wall carbon nanotubes and gold nanorods on a per mass basis. This allows for whole-body lymph node PA mapping with low injection doses. SPNs also possess high structural flexibility, narrow PA spectral profiles, and resistance to photodegradation and oxidation, enabling the development of a NIR ratiometric PA probe for real-time imaging of reactive oxygen species (ROS). The probe demonstrates significant PA signal changes in response to ROS, making it suitable for monitoring pathological processes in vivo. The study highlights the potential of SPNs as a nanoplatform for advanced PA molecular imaging, offering deeper tissue penetration, higher spatial resolution, and the ability to simultaneously acquire anatomical and molecular information.This study introduces semiconducting polymer nanoparticles (SPNs) as a new class of contrast agents for photoacoustic (PA) molecular imaging in living mice. SPNs, derived from photovoltaic semiconducting polymers, exhibit strong NIR light absorption and produce higher PA signals compared to single-wall carbon nanotubes and gold nanorods on a per mass basis. This allows for whole-body lymph node PA mapping with low injection doses. SPNs also possess high structural flexibility, narrow PA spectral profiles, and resistance to photodegradation and oxidation, enabling the development of a NIR ratiometric PA probe for real-time imaging of reactive oxygen species (ROS). The probe demonstrates significant PA signal changes in response to ROS, making it suitable for monitoring pathological processes in vivo. The study highlights the potential of SPNs as a nanoplatform for advanced PA molecular imaging, offering deeper tissue penetration, higher spatial resolution, and the ability to simultaneously acquire anatomical and molecular information.