Herein, NiO nanorods (NiONRs) was synthesized using Mangifera indica leaves extract and decorated with chitosan curcumin analog Schiff base (Cs-Cur-A). The characterization of the NiONRs and Cs-Cur-A were investigated through different techniques including FT-IR, 1HNMR, XRD, SEM and EDX. A sensitive and selective electrochemical sensor to determine 5-Fluorouracil (5-FU) was elucidated using a Cs-Cur-A/NiONRs modified glassy carbon electrode (GCE). The anodic peak current of 5-FU was greatly enhanced at the Cs-Cur-A/NiONRs/GCE with pH 7.0 and a scan rate of 50 mV/s. According to chronoamperometric measurements, the value of diffusion coefficient (D) was estimated to be 2.96 × 10−6 cm2/s. Using differential pulse voltammetry (DPV), the fabricated sensor exhibits a wide linear range (0.1–150.0 μM) with low limit of detection (21.75 nM). Moreover, the proposed sensor was successfully applied to the detection of 5-FU in blood serum and urine samples with high reproducibility, repeatability and selectivity.