Detection of organic pollutants and biomolecules at very low concentrations is a great challenge for protecting the environment and diagnosis of human health. Noble metal nanomaterials have been employed as active surface-enhanced Raman spectroscopy (SERS) substrates for such detection but suffer with high cost. The two-dimensional (2D) transition-metal dichalcogenides (TMDs) can be used as cost-effective alternative SERS substrates for the detection of organic impurities and biomolecules. Here, we report the hydrothermally synthesized in-situ grown MoS2 films over conducting carbon paper (CCP) and fluorine-doped tin oxide (FTO) coated glass and used them as SERS substrates for the detection of Rhodamine 6G (R6G), vitamin B12 and bilirubin. We observe that hydrothermally in-situ grown MoS2 film over FTO coated glass (MoS2-FTO) shows a better SERS detection limit for studied analytes compared to the in-situ grown MoS2 film over conducting carbon paper (MoS2-CCP). We observe the detection of R6G, vitamin B12, and bilirubin with maximum enhancement factors of 2.42 × 106, 4.18 × 102, and 2.78 × 106, respectively, using MoS2-FTO as SERS substrate. Furthermore, we demonstrate the electrochemical-SERS (EC-SERS) activity of prepared SERS substrates. The MoS2-FTO shows a better EC-SERS signal as compared to MoS2-CCP due to the high conductivity (maximum charge transfer at oxidation potential), strong chemical adsorption, change of orientation, and density of the molecules on the surface of the substrate.