In this study, copper sulfide nanostructures (CuS) and CuS-Cu hybrid structures were synthesized and tested for their potential in electrochemical sensing of homocysteine (HcySH). The determination of HcySH assumes high clinical relevance, as it is a biomarker of cardiovascular diseases, and an indicator of vitamin B12 deficiency. We introduce a portable and versatile sensor system by preparing an FTO-based electrode, thus enhancing the practical utility of the CuS-based sensor in diverse applications. The electrochemical sensing activity is tested in GC/CuS-Cu and FTO/CuS-Cu electrodes. The fabricated FTO/CuS-Cu stand-alone electrode system exhibited superior performances in the quantitative estimation of HcySH. The cyclic voltammetric response showed linearity of current against the concentration of HcySH in the range from 0 to 50 μM.The amperometric response of the FTO/CuS-Cu electrode against different concentrations of HcySH varies linearly in the range of 16 to 83 μM. The limit of detection (LOD) of the system for HcySH is 1.63 µM. CuS and Cu NPs form an electrically conductive layer at the surface of the electrode, and they enable active sites and provide direct electron transfer. The synergetic combination of “binding Cu NPs with HcySH” and the “catalytic activity of CuS in the redox reaction of HcySH” might facilitate electrochemical communication. The platform is also studied in Fetal Bovine Serum (FBS), and it can be employed as a low-cost point-of-care diagnostic tool.