© 2020 Wiley Periodicals LLCPhenolic compounds used in food industries and pesticide industry, are environmentally toxic and pollute the rivers and ground water. For that reason, detection of phenolic compounds such as catechol by using simple, efficient and cost-effective devices have been becoming increasingly popular. In this study, a suitable and a novel matrix was composed using a novel conjugated polymer, namely poly[1-(5-(4,8-bis(5-(2-ethylhexyl)thiophen-2-yl)benzo[1,2-b:4,5-b']dithiophen-2-yl)furan-2-yl)-5-(2-ethylhexyl)-3-(furan-2-yl)-4H thieno[3,4-c]pyrrole-4,6(5H)-dione] (PFTBDT) and carbon dots (CDs) to detect catechol. PFTBDT and CDs were synthesized and the optoelectronic properties of PFTBDT were investigated via electrochemical and spectroelectrochemical studies. Laccase enzyme was immobilized onto the constructed film matrix on the graphite electrode. The proposed biosensor was found to have a low detection limit (1.23 μM) and a high sensitivity (737.44 μA/mM.cm−2) with a linear range of 1.25–175 μM. Finally, the applicability of the proposed enzymatic biosensor was evaluated in a tap water sample and a satisfactory recovery (96–104%) was obtained for catechol determination.