In simplicity, bacteriophages (phages in short) are entities with ability to infect bacteria. Phage particles constitute a potential source of alternatives for the practical application (therapy, biotechnology, food safety). Endolysins are phage- encoded lytic enzymes that are responsible for enzymatic degradation of peptidoglycan layer of the host bacterium. Bacterial elimination is not their only unique way how to use them: many studies have led to the knowledge how to possibly bind parts of endolysins to the bacterial cell wall. This understanding can be used in rapid diagnostic methods on various bacterial strains. Bacteriophage endolysins targeting Gram-positive bacteria feature a modular structure and so are composed of at least two separated functional domains: the N-terminal catalytic domain; and the C-terminal domain with the host cell wall binding activity. Cell wall binding domain is responsible for the recognition of specific receptors in the cell wall of bacteria. Connection between endolysins and bacterial cell walls has major impact on the extent of enzyme activity. This ability provides specificity to a particular bacterial strain or species and significantly affects the endolysin efficiency. Therefore, the nucleotide sequence of a universal binding domain (UBD) was designed for endolysin applicable on multiple strains of Staphylococcus aureus and Streptococcus agalactiae strains. These bacteria represent pathogens, causing infectious human and veterinary diseases and contaminating foodstuffs. In the bioinformatics part, extended analyses were performed, comprising cell wall binding domains of endolysins from phages infecting S. agalactiae, S. aureus, in which sequence alignments of SH3 binding domains were assessed. Translations of 3D models were created through the MultiProt server, namely SH3 biding domain of an endolysin from bacteriophage infecting S. agalactiae and of tertiary structure templates, which were identified by the program PHYRE as default in modelling. Subsequently, plasmid construct pET21a-ubd1-gfp was prepared, containing the proposed ubd1 in fusion with the gene for green fluorescent protein (GFP). After the verification of cloning, induced heterogeneous expression of UBD1-GFP fusion protein was carried out in Escherichia coli. UBD1-GFP expressed fusion protein was partially purified by ion affinity chromatography and analyzed for purity by SDS-PAGE. In specific binding assays, UBD1-GFP was incubated either with S. aureus, or with S. agalactiae cells. UBD binding activity to the cells was confirmed by fluorescence microscopy. Along with this, the detection of different S. aureus or S. agalactiae strains was performed, which could also find a potential application in microbiological practise.