The assessment of phenolic compounds in food samples, environmental samples, and medical applications has gained importance recently. Here, we present research on novel conjugated polymer nanoparticles (P-PimBzBt NPs) and their composites with two-dimensional titanium disulfide nanosheets (2D-TiS2) for electrochemical tyrosinase (TYR)-based catechol detection. P-PimBzBt NPs are decorated with 2D-TiS2 to enhance the electrochemical performance for biosensing. In addition, the interaction of P-PimBzBt NPs with TiS2 was investigated at the molecular level by employing van der Waals (vdW) dispersion-corrected density functional theory (DFT) calculations and classical all-atom molecular dynamics simulations. According to the theoretical studies, the presence of the TiS2 layer increases the interfacial interaction with the conjugated polymer via electrostatic interactions. Using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), field emission scanning electron microscopy (FE-SEM), and energy-dispersive X-ray spectroscopy (EDS) analyses, the production of SPE/TiS2@P-PimBzBt NPs/TYR nanobiosensors was examined. With a detection range of 3.0-27.5 μM, 0.33 μM LOD, and 3.89 μA/μM·cm2 sensitivity values, the sensing layer based on the TiS2@P-PimBzBt NP composites has a targeting ability toward catechol. Its selectivity was investigated using commonly used interfering ions and compounds such as citric acid, urea, glucose, uric acid, KCl, and NaCl. Application of nanobiosensors to actual samples (tap water and black tea) was carried out with high accuracy. The fabricated biosensing platform demonstrates that P-PimBzBt NPs with 2D-TiS2 nanomaterial functionalization are appropriate as electrode materials and could be used to create an inexpensive, fast-response, and highly selective electrochemical biosensor for the detection of catechol in actual samples.