Pyocyanin (PYO) is a green blue pigment that is produced extracellularly by the Gram-negative bacteria Pseudomonas aeruginosa. Its color depends on pH value. It exists in blue zwitterion form at neutral and alkaline conditions, while in an acidic environment, it becomes pink after protonation. PYO has shown the antibacterial activity, as well as the ability to inhibit the growth of fungi like Aspergillus fumigatus and Candida albicans. Moreover, it shows the high cytotoxic effect against the human pancreatic cancer cells by inducing their apoptosis. To evaluate the possible mechanism of biological activity of PYO, in the present study, we have investigated its interactions with calf thymus DNA (ct-DNA) and bovine serum albumin (BSA) by fluorescence emission spectroscopy. The obtained value of binding constant to BSA is relatively high (K_A = 5.3 × 10⁶ M^-1s^-1), showing the ability of PYO to bind to this transport protein. We have also used synchronous fluorescence spectroscopy to explore the structural changes in BSA in the presence of the studied biopigment. In contrast with the mentioned results for binding to BSA, PYO has shown a low affinity to ct-DNA, what can be seen from the value of its binding constast (K_A = 7.8 × 10³ M^-1s^-1).