In this work we investigated the effect of a DNA oligonucleotide sequence on the activity of a DNAzyme with covalently attached hemin. For this purpose, we synthesized seven DNA-hemin conjugates. All DNA-hemin conjugates as well as DNA/hemin complexes were characterized using circular dichroism, determination of melting temperatures and pKa of hemin. We observed that hemin conjugation in most cases led to the formation of parallel G-quadruplexes in the presence of potassium and increased thermal stability of all studied systems. Although the activity of DNA-hemin conjugates depended on the sequence used, the highest activity was observed for the DNA-hemin conjugate based on a human telomeric sequence. We used this DNAzyme for development of “sandwich” assay for detection of DNA sequence. For this assay, we used electric chip which could conduct electricity after silver deposition catalyzed by DNAzyme. This method was proved to be selective towards DNA oligonucleotides with mismatches and could be used for the detection of the target. To prove the versatility of our DNAzyme probe we also performed experiments with streptavidin-coated microplates. Our research proved that DNAzyme with covalently attached hemin can be used successfully in the development of heterogeneous assays.