In this work, the possibility of precise physicochemical analysis of formation of nucleic acid complexes has been demonstrated using thermal cyclers with fluorescence signal detection. Individual features of heating blocks of various thermal cyclers have been identified. A nonlinear correlation between fluorescence signal intensity and concentration of fluorescent dyes (5,6)-carboxyfluorescein and Rhodamine B has been identified. Using UV and fluorescence melting, the comparison of melting curves at various concentration of full-complementary and mismatched (A/A and C/A) complexes has been conducted. The experimental values of thermodynamic parameters of complex formation for full-complementary and mismatched complexes have been obtained.