Semiconducting polymer nanoparticles (SPNs) have emerged as an alternative class of optical nanoagents for imaging applications. However, the general preparation method of SPNs is nanoprecipitation, which is likely to encounter the issue of nanoparticle dissociation. We herein report nondissociable near-infrared (NIR)-absorbing organic semiconducting nanoparticles for in vivo photoacoustic (PA) and fluorescence imaging. The nanoparticles are self-assembled from an amphiphilic semiconducting oligomer (ASO) that has a hydrophobic semiconducting oligomer backbone attached by hydrophilic poly(ethylene glycol) (PEG) side chains. The ASO has a higher structural stability and brighter PA signals compared to those of its counterpart nanoparticles synthesized by nanoprecipitation. The small size and the PEG-passivated surface of the ASO allow it to passively target to and efficiently accumulate in the tumor of living mice, permitting tumor imaging with high signal-to-background ratios. Our study provides new NIR-absorbing organic nanoparticles for PA and fluorescence imaging, which also have the potential to be used as a drug carrier for theranostics.