Borylated poly(fluorene-benzothiadiazoles) (PF8-BT) are π-conjugated polymers (CPs) with deep-red/ near-infrared (NIR) absorption and emission profiles suitable for in vivo optical imaging. A fully borylated PF8-BT derivative (P4) was encapsulated in pegylated poly(lactic- co-glycolic acid) (PEG-PLGA) nanoparticles and compared with a reference NIR-emitting CP (PCPDTBT) or indocyanine green (ICG). All formulations satisfied quality requirements for parenterally administered diagnostics. P4 nanoparticles had higher quantum yield (2.3%) than PCPCDTBT (0.01%) or ICG nanoparticles (1.1%). The signal/background ratios (SBRs) of CP systems P4 and PCPDTBT in a phantom mouse (λ em = 820 nm) increased linearly with fluorophore mass (12.5-100 μg/mL), while the SBRs of ICG decreased above 25 μg/mL. P4 nanoparticles experienced <10% photobleaching over 10 irradiations (PCPDTBT: ∼25% and ICG: >44%). In a mouse tumor xenograft model, P4 nanoparticles showed a 5-fold higher SBR than PCPDTBT particles with fluorophore accumulation in the liver > spleen > tumor. Blood chemistry and tissue histology showed no abnormalities compared to untreated animals after a single administration.