This study describes the formation of single‐chain polymer dots (Pdots) via ultrasonic emulsification of nonionic donor‐acceptor‐donor type (D–A–D) alkoxy thiophene–benzobisthiadiazole‐based conjugated polymers (Poly BT) with amphiphilic cetyltrimethylammonium bromide (CTAB). The methodology yields Pdots with a high cationic surface charge (+56.5 mV ± 9.5) and average hydrodynamic radius of 12 nm. Optical characterization reveals that these Pdots emit near‐infrared (NIR) light at a maximum wavelength of 860 nm owing to their conjugated polymer backbone consisting of D–A–D monomers. Both colloidal and optical properties of these Pdots make them promising fluorescence emissive probes for bioimaging applications. The significant advantage of positively charged Pdots is demonstrated in diffusion‐limited mediums such as tissues, utilizing human epithelial breast adenocarcinoma, ATCC HTB‐22 (MCF‐7), human bone marrow neuroblastoma, ATCC CRL‐2266 (SH‐SY5Y), and rat adrenal gland pheochromocytoma, CRL‐1721 (PC‐12) tumor spheroid models. Fluorescence microscopy analysis of tumor spheroids from MCF‐7, SH‐SY5Y, and PC‐12 cell lines reveals the intensity profile of Pdots, confirming extensive penetration into the central regions of the models. Moreover, a comparison with mitochondria staining dye reveals an overlap between the regions stained by Pdots and the dye in all three tumor spheroid models. These results suggest that single‐chain D–A–D type Pdots, cationized via CTAB, exhibit long‐range mean free path of penetration (≈1 µm) in dense mediums and tumors.