The present research utilized a simplified procedure for developing a novel electrochemical sensor based on a carbon paste electrode (CPE) modified with single-stranded DNA (ss-DNA), reduced graphene oxide and molybdenum disulfide. Unmodified (bare CPE) and modified (ss-DNA/RGO/MoS2/CPE) electrodes were characterized by scanning electron microscopy (SEM), EDX analysis and cyclic voltammetry (CV). Characterization data confirm the good conductivity and electrocatalytic nature with more electrochemically active sites in ss-DNA/RGO/MoS2/CPE compared to bare CPE in determination of capecitabine (CAP) analysis in real samples. Two linear ranges have been obtained for the CAP concentration within the ranges of 0.01-10 µM with and 10–60 µM, with a detection limit of 0.0108 µM and limit of quantification of 0.036 µM. Lower linear concentration range, 0.01-10 µM showed sensitivity of 276.85 AM− 1cm− 2, and another from 10 µM to 60 µM with a sensitivity of 5.88 AM− 1cm− 2. The performance of the modified electrode was tested in human serum samples and obtained satisfactory recovery results. The selectivity and practical ability of ss-DNA/RGO/MoS2/CPE to determine CAP in the presence of different interfering species were investigated. The results show the selective, reliable and accurate response of ss-DNA/RGO/MoS2/CPE as CAP electrochemical sensor.