The substrate properties of six pairs of fluorescently labeled deoxyuridine and deoxycytidine triphosphates (Cy5-dUTPs and Cy5-dCTPs) in PCR with Taq polymerase were compared. In each pair, the modified dU and dC contained identical fluorescently labeled Cy5 substituents; for different pairs, the sub-stituent structures differed in the length of the linker between the nitrogenous base and the fluorophore, the length of the linker between the quaternary ammonium group and the second heterocycle of the fluorophore, as well as the structure of the fluorophore itself. DNA fragments of Staphylococcus aureus (AT-rich template) and Mycobacterium tuberculosis (GC-rich) were used as templates. With both templates, deoxycytidine derivatives showed slightly higher amplification efficiency (E). The influence of the fluorophore structure and the GC-composition of the template on the kinetics of the reaction was insignificant. At the same time, a high incorporation efficiency was observed on the AT-rich template for uridine derivatives, and on the GC-rich template for cytidine derivatives (and in both cases, for substituents with a longer linker length). Nevertheless, the specific incorporation density, which takes into account the number of similar nucleotides in the DNA chain, was in all cases higher for dU derivatives. It was found that in pairs with similar fluorophore modifications, uridine derivatives, compared with cytidine, are characterized by a higher incorporation density, regardless of the composition of the template, but at the same time they have a greater inhibitory effect. The results obtained will increase the sensitivity of fluorescence analysis using the immobilized phase (microarray analysis).