Bioconjugation, biosensing, bioimaging, bionanomaterials, etc., are only a few examples of application of functionalized DNA. Since base-modified nucleic acids contribute not only to a broad range of biotechnological fields but also to the understanding of various cellular processes, it is crucial to design novel modifications with unique properties. Here, we demonstrate the utilization of N4-cytidine modified oligonucleotides, which contain reactive acetophenone (AP) or benzophenone (BP) groups, for the UV-induced cross-linking. We find that terminal deoxynucleotidyl transferase-mediated 3'-tailing using AP/BP-containing modified nucleotides generates photoactive DNA, suitable for a straightforward covalent cross-linking with both interacting proteins and a variety of well-known solid polymeric supports. Moreover, we show that AP/BP-functionalization of nucleic acid molecules induces an efficient cross-linking upon exposure to UVA light. Our findings reveal that 3'-tailed single-stranded DNA bearing AP/BP-moieties is easily photoimmobilized onto untreated polystyrene, polypropylene, polylactate, polydimethylsiloxane, sol-gel and borosilicate glass substrates. Furthermore, we demonstrate that such immobilized DNA probes can be further used for successful hybridization of complementary DNA targets. Our results establish novel N4-cytosine nucleobase modifications as photoreactive labels and suggest an effortless approach for photoimmobilization of nucleic acids.