The network-like cobalt oxide was electrochemically prepared on the nickel oxide nanoworms (NiONWs) which were created on the surface of a nickel film (NF) substrate. Firstly, a very thin layer of NiO nanoworms was successfully grown on the anodized Ni foil. Thereafter, the 3D Co3O4 nanosheets were directly electrodeposited on a-NF/NiONWs by using cyclic voltammetry. The prepared NF/NiONWs/Co3O4 was used as a sorbent for the thin film microextraction (TFME) of diclofenac as the model compound in biological fluids for the first time. The extracted diclofenac was then analyzed by high performance liquid chromatography-ultraviolet detection (HPLC-UV). A Plackett–Burman design was performed for screening the experimental factors to specify the significant variables affecting the performance of the method. The effective factors were then optimized using Box-Behnken design (BBD). Under the optimum condition, the limit of detection (LOD) in water matrix was 0.15 μg L−1. Limit of quantification (LOQ) was also 0.58 μg L−1. Relative standard deviation for the analyte extraction at the spiked concentration of 5 μg L−1 was 4.7% (as intra-day RSD). Film to film reproducibility at the spiked concentration of 100 μg L−1 was also 6.8%. The linearity of the method in water, urine and plasma matrix was in the range of 1–200, 1–200 and 2–100 μg L−1, respectively. The capability of the film was investigated by extraction and determination of diclofenac in different biological fluids including urine and plasma samples.
