This study introduces near-infrared (NIR) light-absorbing semiconducting polymer nanoparticles (SPNs) as a new class of contrast agents for photoacoustic (PA) molecular imaging in living mice. SPNs offer stronger signal output than commonly used single-wall carbon nanotubes (SWNTs) and gold nanorods (GNRs) on a per mass basis, enabling whole-body lymph node PA mapping in living mice with low systemic injection mass. SPNs exhibit high structural flexibility, narrow PA spectral profiles, and strong resistance to photodegradation and oxidation, making them ideal for developing the first NIR ratiometric PA probe for in vivo real-time imaging of reactive oxygen species (ROS), which are vital chemical mediators in many diseases. SPNs are synthesized from photovoltaic semiconducting polymers (SPs) with strong NIR absorption. Two SP derivatives, SP1 and SP2, were used to prepare SPNs through nanoprecipitation with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), resulting in water-soluble nanoparticles with sizes around 40 nm. SPNs showed high NIR absorption with maxima at 660 and 700 nm for SP1 and SP2, respectively. SPN1 exhibited a higher peak mass extinction coefficient than SPN2, making it a better platform for PA molecular imaging. SPNs were compared with SWNT and GNR in agar phantoms and living mice. SPN1 showed significantly higher PA amplitude than SWNT and GNR at the same mass concentration. SPN1 also demonstrated superior photostability and suitability for long-term PA molecular imaging. SPN1 was effective for PA imaging of lymph nodes (LN), showing high accumulation in LN and a 13.3-fold enhancement in PA signal following SPN administration. SPNs were further developed into an activatable NIR ratiometric PA probe (RSPN) for ROS imaging. RSPN was designed by coupling SPN1 with a cyanine dye derivative (IR775S) sensitive to ROS-mediated oxidation. RSPN showed a dual-peak ratiometric PA response to different ROS, with a PA ratio increasing significantly in the presence of ONOO⁻ and ClO⁻. RSPN effectively detected in vivo ROS production using a dual-peak ratiometric PA contrast mechanism, demonstrating the potential of SPNs for activatable PA imaging of pathological processes in real time. SPNs offer advantages such as high photostability, narrow PA spectral profiles, and the ability to be formulated with distinct PA wavelengths without altering pharmacokinetic profiles. They can be used for both simple accumulation-based imaging and as a nanoplatform for activatable probes. Given their many key merits, SPNs show great promise for advanced PA molecular imaging, from preclinical investigation of physiological and pathological processes to enhancing theThis study introduces near-infrared (NIR) light-absorbing semiconducting polymer nanoparticles (SPNs) as a new class of contrast agents for photoacoustic (PA) molecular imaging in living mice. SPNs offer stronger signal output than commonly used single-wall carbon nanotubes (SWNTs) and gold nanorods (GNRs) on a per mass basis, enabling whole-body lymph node PA mapping in living mice with low systemic injection mass. SPNs exhibit high structural flexibility, narrow PA spectral profiles, and strong resistance to photodegradation and oxidation, making them ideal for developing the first NIR ratiometric PA probe for in vivo real-time imaging of reactive oxygen species (ROS), which are vital chemical mediators in many diseases. SPNs are synthesized from photovoltaic semiconducting polymers (SPs) with strong NIR absorption. Two SP derivatives, SP1 and SP2, were used to prepare SPNs through nanoprecipitation with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), resulting in water-soluble nanoparticles with sizes around 40 nm. SPNs showed high NIR absorption with maxima at 660 and 700 nm for SP1 and SP2, respectively. SPN1 exhibited a higher peak mass extinction coefficient than SPN2, making it a better platform for PA molecular imaging. SPNs were compared with SWNT and GNR in agar phantoms and living mice. SPN1 showed significantly higher PA amplitude than SWNT and GNR at the same mass concentration. SPN1 also demonstrated superior photostability and suitability for long-term PA molecular imaging. SPN1 was effective for PA imaging of lymph nodes (LN), showing high accumulation in LN and a 13.3-fold enhancement in PA signal following SPN administration. SPNs were further developed into an activatable NIR ratiometric PA probe (RSPN) for ROS imaging. RSPN was designed by coupling SPN1 with a cyanine dye derivative (IR775S) sensitive to ROS-mediated oxidation. RSPN showed a dual-peak ratiometric PA response to different ROS, with a PA ratio increasing significantly in the presence of ONOO⁻ and ClO⁻. RSPN effectively detected in vivo ROS production using a dual-peak ratiometric PA contrast mechanism, demonstrating the potential of SPNs for activatable PA imaging of pathological processes in real time. SPNs offer advantages such as high photostability, narrow PA spectral profiles, and the ability to be formulated with distinct PA wavelengths without altering pharmacokinetic profiles. They can be used for both simple accumulation-based imaging and as a nanoplatform for activatable probes. Given their many key merits, SPNs show great promise for advanced PA molecular imaging, from preclinical investigation of physiological and pathological processes to enhancing the