The influence of methyl-, 2-hydroxyethyl-, dimethyl-, diethyl- and benzoyl-amino substituents in the 6,6'-positions of benzothiazole heterocycle of trimethine cyanines on their spectral-luminescent properties and behavior in presence of DNA, RNA and BSA was studied. It was shown that incorporation of 6,6'-substituents generally leads to the increase in dyes tendency to aggregation, resulting in the considerable decrease in the emission intensity of the disubstituted dyes as compared to the unsubstituted ones. Emission of the studied 6,6'-disubstited dyes in DNA presence is considerably more intensive than in presence of RNA, that points on the existing of DNA binding preference for the mentioned dyes. Insertion of benzoyl-amino groups into the 6,6'-positions permitted us to design the DNA-sensitive dyes on the basis of symmetric trimethine cyanines with unsubstituted polymethine chain, while typically such dyes slightly respond on the presence of biopolymers. 6,6'-Benzoyl-amino-disubstituted trimethine cyanines are proposed as efficient dyes for DNA detection.