The spectral characteristics of monosubstituted derivatives of 2,2'-bithiophene (1-BT) with simple carbonyl substituents, including -COCF3 (trifluoroacetyl, 2-BTCF), -COCH2CN (oxopropanenitrile, 3-BTCN) and -COCH3 (acetyl, 4-BTCE), and the more complex 5'-((9H-fluoren-9-ylidene)methyl)-3-methyl, 5-methyl carboxylate (5-BTFL) were investigated. Absorption and fluorescence spectra and fluorescence lifetimes were measured in solvents with various polarities and compared with those in polymer matrices (polystyrene, PS; polymethyl methacrylate, PMMA; and polyvinyl chloride, PVC). Although the parent, 1-BT, absorbed near 300 nm and exhibited no or weak fluorescence, the substitution of 1-BTwith simple substituents at position 5 resulted in a bathochromic shift of approximately 50 nm or more in absorption and distinct fluorescence above 400 nm. The largest shift in absorption and fluorescence was observed for the complex 5-BTFL with fluorene as a substituent for 1-BT. The most intense fluorescence was observed for the derivative 2-BTCF with trifluoroacetyl as a substituent in the polar PVC matrix. The lifetimes of fluorescence of all substituted 2,2'-bithiophenes were in the range from 0.3 to 3 ns. The polymer matrices increased the intensity of fluorescence to some extent and prolonged the lifetime of the 2,2'-bithiophene derivatives. The reasons for the variation in the fluorescence intensity resulting from the substitution of the parent dimer are discussed.