A comparative study of the optical properties of organic fluorescent nanoparticles fabricated by laser ablation (NPs-LA), reprecipitation (NPs-RP), and microemulsion (NPs-ME) methods is presented. These nanoparticles contain a fluorene-based p-conjugated molecule (BT2). Distinctive electronic transitions are observed in samples due to the specific way in which the molecule BT2 is assembled in each type of nanoparticles; for instance, transitions involved in absorption and emission spectra of NPs-LA result in blueshifting with respect to the molecular solution of BT2, whereas redshifting is observed in NPs-RP and NPs-ME. Further, the results show that under infrared excitation, the aqueous suspensions of NPs-LA exhibit the highest fluorescence induced by two-photon absorption (approximate to 790 GM at 740 nm), as well as the best photostability, compared with aqueous suspensions of NPs-RP and NPs-ME. The nanoparticles synthetized by the three aforementioned methods are employed as exogenous agents for the visualization of human cervical cancer cell line (HeLa) using confocal and two-photon microscopy. Under similar experimental conditions, it is found that microscopy images of the best quality are obtained with NPs-LA. These results show that laser ablation is a suitable technique for the fabrication of organic fluorescent nanoparticles used as contrast agents for in vitro fluorescence microscopy.