A strong and stable fluorescent signal is the crux for the ultrasensitive biodetection technology, conjugated polymer nanoparticles (CPNs) as new fluorescence labels have attracted more and more attention for their excellent optical properties. However, a systematic understanding of the size-dependent optical properties of CPNs with diameters from the nano to submicron ranges is lacking, which is the most important issue when choosing label materials. Hence, poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1 0 ,3}-thiadiazole)] (PFBT) nanoparticles with sizes from 50 nm to 200 nm were synthesized and studied. It was demonstrated that the spectroscopic and fluorescent properties are similar for CPNs with different sizes.The relationship between single-particle brightness and diameter was investigated via fluorescence spectrometry and fluorescence correlation spectroscopy (FCS), and the results presented that the singleparticle brightness increased quadratically with the increase of the diameter of CPNs. This research may provide valuable support to further application of CPNs in biological diagnostics.